SummaryThe plastid encoded RNA polymerase subunit genes rpoA, B and C1 of tobacco were disrupted individually by PEG-mediated plastid transformation. The resulting off-white mutant phenotype is identical for inactivation of the different genes. The mutants pass through a normal ontogenetic cycle including¯ower formation and production of fertile seeds. Their plastids reveal a poorly developed internal membrane system consisting of large vesicles and, occasionally,¯attened membranes, reminiscent of stacked thylakoids. The rpo ± material is capable of synthesising pigments and lipids, similar in composition but at lower amounts than the wild-type. Western analysis demonstrates that plastids contain nuclear-coded stroma and thylakoid polypeptides including terminally processed lumenal components of the Sec but not of the DpH thylakoid translocation machineries. Components using the latter route accumulate as intermediates. In striking contrast, polypeptides involved in photosynthesis encoded by plastid genes could not be detected by Western analysis, although transcription of plastid genes, including the rrn operon, by the plastid RNA polymerase of nuclear origin is found as expected. Remarkably, ultrastructural, sedimentation and Northern analyses as well as pulse experiments suggest that rpo ± plastids contain functional ribosomes. The detection of the plastidencoded ribosomal protein Rpl2 is consistent with these results. The ®ndings demonstrate that the consequences of rpo gene disruption, and implicitly the integration of the two plastid polymerase types into the entire cellular context, are considerably more complex than presently assumed.

Plastids contain a NAD(P)H-plastoquinone-oxidoreductase (NDH complex) which is homologous to the eubacterial and mitochondrial NADH-ubiquinone-oxidoreductase (complex I), but the metabolic function of the enzyme is unknown. The enzyme consists of at least eleven subunits (A-K), which are all encoded on the plastid chromosome. We have mutagenized ndhC and ndhJ by insertion, and ndhK and ndhA-I by deletion and insertion, of a cassette which carried a spectinomycin resistance gene as a marker. The transformation was carried out by the polyethylene glycol-mediated plastid transformation method. Southern analysis revealed that even after repeated regeneration cycles each of the four different types of transformants had retained 1-5% of wild-type gene copies. This suggests that complete deletion of ndh genes is not compatible with viability. The transformants displayed two characteristic phenotypes: (i) they lack the rapid rise in chlorophyll fluorescence in the dark after illumination with actinic light for 5 min; in the wild-type this dark-rise reflects a transient reduction of the plastoquinone pool by reduction equivalents generated in the stroma; and (ii) transformants with defects in the ndhC-K-J operon accumulate starch, indicating inefficient oxidation of glucose via glycolysis and the oxidative pentose phosphate pathway. Both observations support the theory of chlororespiration, which postulates that the NDH complex acts as a valve to remove excess reduction equivalents in the chloroplast.

Maca, Lepidium meyenii Walpers (Brassicaceae), is an annual herbaceous plant native to the high plateaus of the Peruvian central Andes. Its underground storage hypocotyls have been a traditional medicinal agent and dietary staple since pre-Columbian times. Reported properties include energizing and fertility-enhancing effects. Published reports have focused on the benzylalkamides (macamides) present in dry hypocotyls as one of the main bioactive components. Macamides are secondary amides formed by benzylamine and a fatty acid moiety, with varying hydrocarbon chain lengths and degree of unsaturation. Although it has been assumed that they are usually present in fresh undamaged tissues, analyses show them to be essentially absent from them. However, hypocotyls dried by traditional Andean postharvest practices or industrial oven drying contain up to 800μgg(-1) dry wt (2.3μmolg(-1) dry wt) of macamides. In this study, the generation of macamides and their putative precursors were studied during nine-week traditional drying trials at 4200m altitude and in ovens under laboratory conditions. Freeze-thaw cycles in the open field during drying result in tissue maceration and release of free fatty acids from storage and membrane lipids up to levels of 1200μgg(-1) dry wt (4.3μmolg(-1) dry wt). Endogenous metabolism of the isothiocyanates generated from glucosinolate hydrolysis during drying results in maximal benzylamine values of 4300μgg(-1) dry wt (40.2μmolg(-1) dry wt). Pearson correlation coefficients of the accumulation profiles of benzylamine and free fatty acid to that of macamides showed good values of 0.898 and 0.934, respectively, suggesting that both provide sufficient substrate for amide synthesis during the drying process.

Plastids of higher plants operate with at least two distinct DNA-dependent RNA polymerases, which are encoded in the organelle (PEP) and in the nucleus (NEP), respectively. Plastid run-on assays and Northern analyses were employed to analyse gene expression in tobacco mutant plastids lacking the PEP genes rpoA, rpoB or rpoC1. Hybridisation of run-on transcripts to restriction fragments representing the entire tobacco plastid chromosome, as well as to selected plastid gene-specific probes, shows that all parts of the plastid DNA are transcribed in rpo-deficient plastids. In comparison to wild-type chloroplasts, which are characterized by preferential transcription of photosynthesis-related genes in the light, mutant plastids exhibit a different transcription pattern with less pronounced differences in the hybridisation intensities between the individual genes. The analysis of steady-state transcript patterns and transcription rates of selected genes in both types of plastids demonstrates that differences in transcription rates are not necessarily paralleled by corresponding changes in transcript levels. The accumulation of large transcripts in the mutant plastids indicates that processing of primary transcripts may be impaired in the absence of PEP. These data suggest that, contrary to the prevailing view, much of the regulation of NEP-driven plastid gene expression in the rpo-deficient mutants is not based on differential promoter usage but is exerted at post-transcriptional levels.

To investigate the influence of mitochondrial genes on stamen development of higher plants, protoplasts from three different, male-sterile tobacco cultivars were fused. The fused cells were cultured individually into calli, from which plants were regenerated. Cybrid plants were obtained that exhibited flowers with recombined biparental malesterile morphology and with novel male-sterile stamens that differed from any types from sexual or somatic hybridizations described previously. The male-sterile morphologies of these cybrids and their parents support the hypothesis that nuclear-mitochondrial interaction occurs at several stages in tobacco floral development and that several mitochondrial genes are necessary for normal stamen and corolla development. Analysis by restriction endonuclease digestion of mitochondrial DNA of male-sterile cybrids and their parents revealed that the mitochondrial DNA of male-sterile cybrids with parental floral morphology was unchanged when compared with parental mitochondrial DNA. Cybrids that were morphologically similar to one parent's male-sterile phenotype had mitochondrial DNA almost identical to that parent, whereas cybrids with recombined biparental or novel male-sterile phenotypes contained mitochondrial DNA different from both male-sterile parents and from each other. A set of mitochondrial DNA fragments could be correlated with split corollas, a feature found in several tobacco male-sterile cultivars. DNA gel blot analysis using a number of mitochondrial genes confirmed the conclusions based on ethidium bromide staining of mitochondrial DNA restriction digests.

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To investigate the influence of mitochondrial genes on stamen development of higher plants, protoplasts from three different, male-sterile tobacco cultivars were fused. The fused cells were cultured individually into calli, from which plants were regenerated. Cybrid plants were obtained that exhibited flowers with recombined biparental malesterile morphology and with novel male-sterile stamens that differed from any types from sexual or somatic hybridizations described previously. The male-sterile morphologies of these cybrids and their parents support the hypothesis that nuclear-mitochondrial interaction occurs at several stages in tobacco floral development and that several mitochondrial genes are necessary for normal stamen and corolla development. Analysis by restriction endonuclease digestion of mitochondrial DNA of male-sterile cybrids and their parents revealed that the mitochondrial DNA of male-sterile cybrids with parental floral morphology was unchanged when compared with parental mitochondrial DNA. Cybrids that were morphologically similar to one parent's male-sterile phenotype had mitochondrial DNA almost identical to that parent, whereas cybrids with recombined biparental or novel male-sterile phenotypes contained mitochondrial DNA different from both male-sterile parents and from each other. A set of mitochondrial DNA fragments could be correlated with split corollas, a feature found in several tobacco male-sterile cultivars. DNA gel blot analysis using a number of mitochondrial genes confirmed the conclusions based on ethidium bromide staining of mitochondrial DNA restriction digests.