MUTAGENESIS OF THE GENES ENCODING SUBUNIT-A, SUBUNIT-C, SUBUNIT-H, SUBUNIT-I, SUBUNIT-J AND SUBUNIT-K OF THE PLASTID NAD(P)H-PLASTOQUINONE-OXIDOREDUCTASE IN TOBACCO BY POLYETHYLENE GLYCOL-MEDIATED PLASTOME TRANSFORMATION

Plastids contain a NAD(P)H-plastoquinone-oxidoreductase (NDH complex) which is homologous to the eubacterial and mitochondrial NADH-ubiquino ne-oxidoreductase (complex I), but the metabolic function of the enzym e is unknown. The enzyme consists of at least eleven subunits (A-K), w hich are all encoded on the plastid chromosome. We have mutagenized nd hC and ndhJ by insertion, and ndhK and ndhA-I by deletion and insertio n, of a cassette which carried a spectinomycin resistance gene as a ma rker. The transformation was carried out by the polyethylene glycol-me diated plastid transformation method. Southern analysis revealed that even after repeated regeneration cycles each of the four different typ es of transformants had retained 1-5% of wild-type gene copies. This s uggests that complete deletion of ndh genes is not compatible with via bility. The transformants displayed two characteristic phenotypes: (i) they lack the rapid rise in chlorophyll fluorescence in the dark afte r illumination with actinic light for 5 min; in the wild-type this dar k-rise reflects a transient reduction of the plastoquinone pool by red uction equivalents generated in the stroma; and (ii) transformants wit h defects in the ndhC-K-J operon accumulate starch, indicating ineffic ient oxidation of glucose via glycolysis and the oxidative pentose pho sphate pathway. Both observations support the theory of chlororespirat ion, which postulates that the NDH complex acts as a valve to remove e xcess reduction equivalents in the chloroplast.