Key words: Chloroplast trans for ma tion; Homol o gous re com bi na tion; Pho to syn the sis; Tobacco; Ycf5. Ab stractHy po thet i cal chloroplast read ing frame 5 (ycf5) is en coded by the chloroplast ge nome and po ten tially spec i fies a pro tein of 313 amino ac ids in to bacco. A north ern blot anal y sis showed that ycf5 tran scripts ac cu mu lated at sub stan tial lev els in the chloroplasts, but not in the proplastids of nonphotosynthetic cul tured cells. In an at tempt to de ter mine the func tion of ycf5, we con structed mu tant al leles for the tar geted de le tion of ycf5. The mu tant al lele was in tro duced into the to bacco chloroplast ge nome by biolistic chloroplast trans for ma tion to re place the cor re spond ing wild-type ycf5 al leles by ho mol o gous re com bina tion. Homoplasmic ycf5-knock out to bacco plants dis played a pale-green phe no type. An anal y sis of the tran sient in crease in chlo ro phyll flu o res cence sug gested that the elec tron flow around photosystem II (PSII) was com pletely im paired in the ycf5-de fi cient transformants. These find ings in di cate that ycf5 is in deed a func tional gene, and that its gene prod uct is involved in the gen er a tion of func tional PSII units. This is the first re port of the iso la tion and char ac ter iza tion of ycf5-knockout mu tants in higher plants.List of ab bre vi a tions: aadA, aminoglycoside 3′′-adenylyltransferase gene; PCR, poly mer ase chain re ac tion; RT-PCR, re verse tran scriptase-PCR; spec, spectinomycin; ycf, hy po thet i cal chloroplast read ing frame. In tro duc tionThe con ser va tive open read ing frames found in land plants and green al gal chloroplast genomes are re ferred to as ycfs (hypothetical chloroplast read ing frame) (Hallick and Bairoch 1994). Based on the amino acid se quence homology to known pro teins of bac te rial and yeast genes, ycf10, ycf11, and ycf14 have been iden ti fied as cemA (Katoh et al. 1996, Rolland et al. 1997, accD (Li and Cronan 1992), and matK (Karsten and Link 1995), re spec tively. The func tions of sev eral ycfs have been as signed by gene-tar geted in ac ti va tion and its bio chem i cal char ac ter iza tion (Rochaix 1997). For in stance, ycf6, ycf7, ycf8, and ycf9 have been des ig nated petN 365 ACTA PHYSIOLOGIAE PLANTARUM

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