MUTATIONS OF CYTOCHROME B(6) IN CHLAMYDOMONAS-REINHARDTII DISCLOSE THE FUNCTIONAL-SIGNIFICANCE FOR A PROLINE TO LEUCINE CONVERSION BY PETB EDITING IN MAIZE AND TOBACCO

We have introduced a proline codon in place of a leucine codon at posi tion 204 of the petB gene of Chlamydomonas reinhardtii. This gene modi fication mimics the presence of proline codons at the same position in the petB genes of maize and tobacco, which are subsequently edited to leucine codons at the RNA level. Following transformation, we observe d no editing at this position in C. reinhardtii, independent of the ty pe of proline codon we have used: the CCA codon, edited in maize, or a CCT codon. Strains carrying the introduced mutation were non phototro phic and displayed a block in photosynthetic electron transfer, consis tent with a lack of cytochrome b(6)f activity. Thus the presence of a proline residue at position 204 in cytochrome b(6) is detrimental ro p hotosynthesis. Wt show that the mutant phenotype arose from a defectiv e assembly of cytochrome b(6)f complexes and not from altered electron transfer properties in the assembled protein complex. Biochemical com parison of the proline-containing transformants with a cytochrome b(6) mutant deficient in heme-attachment indicates that their primary defe ct is at the level of assembly of apocytochrome b(6) with the b(h) hem e, thereby preventing assembly of the whole cytochrome b(6)f complex.