Protein synthesis in chloroplasts. Characteristics and products of protein synthesis in vitro in etioplasts and developing chloroplasts from pea leaves.

The function of plastid ribosomes in pea (Pisum sativum L.) was investigated by characterizing the products of protein synthesis in vitro in plastids isolated at different stages during the transition from etioplast to chloroplast. Etioplasts and plastids isolated after 24, 48 and 96h of greening in continuous white light, use added ATP to incorporate labelled amino acids into protein. Plastids isolated from greening leaves can also use light as the source of energy for protein synthesis. The labelled polypeptides synthesized in isolated plastids were analysed by electrophoresis in sodium dodecyl sulphate-ureapolyacrylamide gels. Six polypeptides are synthesized in etioplasts with ATP as energy source. Only one of these polypeptides is present in a 150 000g supernatant fraction. This polypeptide has been identified as the large subunit of Fraction I protein (3-phospho-D-glycerate carboxylyase EC 4.1.1.39) by comparing the tryptic 'map' of its L-(35S)methionine-labelled peptides with the tryptic 'map' of large subunit peptides from Fraction I labelled with L-(35S)methionine in vivo. The same gel pattern of six polypeptides is seen when plastids isolated from greening leaves are incubated with either added ATP or light as the energy source. However, the rates of synthesis of particular polypeptides are different in plastids isolated at different stages of the etioplast to chloroplast transition. The results support the idea that plastid ribosomes synthesize only a small number of proteins, and that the number and molecular weight of these proteins does not alter during the formation of chloroplasts from etioplasts.     

Identification of the Ndh (NAD(P)H-plastoquinone-oxidoreductase) complex in etioplast membranes of barley: changes during photomorphogenesis of chloroplasts

In the last few years the presence in thylakoid membranes of chloroplasts of a NAD(P)H-plastoquinone oxidoreductase complex (Ndh complex) homologous to mitochondrial complex I has been well established. Herein, we report the identification of the Ndh complex in barley etioplast membranes. Two plastid DNA-encoded polypeptides of the Ndh complex (NDH-A and NDH-F) were relatively more abundant in etioplast membranes than in thylakoids from greening chloroplasts. Conversion of etioplast into chloroplast, after light exposure of barley seedlings grown in the dark, was accompanied by a decrease in the NADH dehydrogenase activity associated to plastid membranes. Using native-PAGE and immunolabelling techniques we have determined that a NADH specific dehydrogenase activity associated with plastid membranes, which was more active in etioplasts than in greening chloroplasts, contained the NDH-A and NDH-F polypeptides. These results complemented by those obtained through blue-native-PAGE indicated that NDH-A and NDH-F polypeptides are part of a 580 kDa NADH dependent dehydrogenase complex present in etioplast membranes. This finding proves that accumulation of the Ndh complex is independent of light. The decrease in the relative levels and specific activity of this complex during the transition from etioplast to chloroplasts was accompanied by a parallel decrease in the specific activity of peroxidase associated to plastid membranes. Based on the mentioned observations it is proposed that an electron transport chain from NADH to H2O2 could be active in barley etioplasts.     

Intercalation of psoralen into DNA of plastid chromosomes decreases late during barley chloroplast development.

We have used a DNA crosslinking assay to measure intercalation of the psoralen derivative HMT (4'-hydroxymethyl-4,5',8-trimethylpsoralen) into barley (Hordeum vulgare) plastid chromosomal DNA during chloroplast and etioplast development. Intercalation into DNA in intact plastids in vivo and in plastid lysates in vitro shows that chromosomal DNA in the most mature chloroplasts intercalates HMT less efficiently than DNA in younger chloroplasts. In contrast, there is no change in HMT intercalation during etioplast differentiation in the dark. Our results also show that DNA in higher plant plastid chromosomes is under superhelical tension in vivo. The lower susceptibility to HMT intercalation of DNA in the most mature chloroplasts indicates that late during chloroplast development the superhelical tension or the binding of proteins to the DNA or both change.     

Evaluation of the Extent of Homologous Chloroplast DNA Sequences in the Mitochondrial Genome of Cowpea (Vigna unguiculata L.)

Southern blot hybridization techniques were used to estimate the extent of chloroplast DNA sequences present in the mitochondrial genome of cowpea (Vigna unguiculata L.) The entire mitochondrial chromosome was homogeneously labeled and used to probe blotted DNA fragments obtained by extensive restriction of the tobacco chloroplast genome. The strongest cross-homologies were obtained with fragments derived from the inverted repeat and the atpBE cluster regions, although most of the clones tested (spanning 85% of the tobacco plastid genome) hybridized to mitochondrial DNA. Homologous chloroplast DNA restriction fragments represent a total of 30 to 68 kilobase pairs, depending upon the presence or absence of tRNA-encoding fragments. Plastid genes showing homology with mitochondrial DNA include those encoding ribosomal proteins, RNA polymerase, subunits of photosynthetic complexes, and the two major rRNAs.     

Architecture of the replication complex and DNA loops at the fork generated by the bacteriophage t4 proteins

Rolling circle replication has previously been reconstituted in vitro using M13 duplex circles containing preformed forks and the 10 purified T4 bacteriophage replication proteins. Leading and lagging strand synthesis in these reactions is coupled and the size of the Okazaki fragments produced is typical of those generated in T4 infections. In this study the structure of the DNAs and DNA-protein complexes engaged in these in vitro reactions has been examined by electron microscopy. Following deproteinization, circular duplex templates with linear tails as great as 100 kb are observed. The tails are fully duplex except for one to three single-stranded DNA segments close to the fork. This pattern reflects Okazaki fragments stopped at different stages in their synthesis. Examination of the DNA-protein complexes in these reactions reveals M13 duplex circles in which 64% contain a single large protein mass (replication complex) and a linear duplex tail. In 56% of the replicating molecules with a tail there is at least one fully duplex loop at the replication complex resulting from the portion of the lagging strand engaged in Okazaki fragment synthesis folding back to the replisome. The single-stranded DNA segments at the fork bound by gene 32 and 59 proteins are not extended but rather appear organized into highly compact structures ("bobbins"). These bobbins constitute a major portion of the mass of the full replication complex.     

Events surrounding the early development of Euglena chloroplasts. 16. Plastid thylakoid polypeptides during greening.

Using sulfolipid to locate plastid thylakoid membranes in gradients from dark-grown resting cells it has been possible to study the plastid thylakoid membrane polypeptides of Euglena gracilis var. bacillaris undergoing light-induced chloroplast development. All plastid thylakoid bands seen in dark-growing wild-type cells and in mutant W3BUL in which plastid DNA is undetectable, are observed to increase in amount during plastid development. Others, which are undetectable in dark-grown wild-type and W3BUL increase greatly during plastid development and appear to be those associated with pigment-protein complexes. The data obtained from experiments where the polypeptides were labeled with 35S during development, either continuously or in pulses, were consistent with these findings. Cycloheximide strongly inhibited the increases in amount in all bands and chloramphenicol or streptomycin produced a lower level of inhibition in all bands indicating tight control of theformation of each plastid membrane constituent by the others. The formation of a polypeptide band of 25 000 molecular weight, thought to be a part of a pigment-protein complex of the thylakoid, and chlorophyll synthesis were inhibited identically by these antibiotics.