Chloroplast Cytochrome b₆/f and ATP Synthase Complexes in Tobacco: Transformation With Antisense RNA Against Nuclear-Encoded Transcripts for the Rieske FeS and ATPδ Polypeptides

Abstract

Tobacco plants with reduced amounts and activities of both the chloroplast cytochrome b₆/f and ATP synthase complexes have been produced using antisense RNA techniques. Antisense constructs were generated from tobacco cDNA clones coding for the Rieske FeS protein and the δ subunit of the b₆/f and ATP synthase complexes respectively. Transformants with altered activities were selected using pulse-modulated fluorescence measurements. The b6/ftransformants showed high levels of steady-state fluorescence and reduced levels of both photochemical and non-photochemical quenching. In striking contrast, the ATP synthase transformants showed low levels of steady-state fluorescence and greatly increased levels of non-photochemical quenching. Transformants with a range of suppression were isolated for both constructs, in some cases with photosynthesis reduced to less than 10% of wild-type values. The most severely affected transformants showed extremely slow growth and in some cases they were unable to grow and produce seed. Progeny from the R1 seed from several cytochrome b₆/f transformants have been analysed and show segregation of phenotypes ranging from intermediate to severe in repression. Intermediate and severe phenotype plants showed a reduction in Rieske FeS mRNA of more than 90% while FeS polypeptide was reduced to 60 and 86% of wild type. There was a strong correlation between photosynthesis at air and Rieske FeS polypeptide content in the antisense plants suggesting that the cytochrome b₆/f complex was a major determinant of photosynthetic rate under these conditions. Photoinhibition studies of FeS antisense plants showed that there was a reduced activity of the xanthophyll cycle in the most severe plants, consistent with a reduction in the transthylakoid pH gradient and a lowered non-photochemical quenching. Preliminary studies of the ATPδ antisense plants showed that they also had reduced levels of mRNA and ATPδ polypeptide.