Phytochrome control of plastid mRNA in mustard (Sinapis alba L.)

The steady-state levels of plastid RNA sequences in dark-grown and light-grown mustard (Sinapis alba L.) seedlings have been compared. Total cellular RNAs were labeled in vitro with ³²P and hybridized to separated restriction fragments of plastid DNA. Cloned DNA fragments which encode the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] and a 35,000 plastid polypeptide were used as probes to assess the levels of these two plastid mRNAs. The 1.22-kilobase-pair mRNA for the 35,000 polypeptide is almost undetectable in dark-grown seedlings, but is a major plastid mRNA in light-grown seedlings. The hybridization analysis of RNA from seedlings which were irradiated with red and far-red light indicates that the level of this mRNA, but not of LS mRNA, is controlled by phytochrome.Abbreviations LS large subunit - RuBP ribulose-1,5-bisphosphate - ptDNA plastid DNA     

Physical and gene mapping of chloroplast DNA from Atriplex triangularis and Cucumis sativa.

A rapid and simple method for constructing restriction maps of large DNAs (100-200 kb) is presented. The utility of this method is illustrated by mapping the Sal I, Sac I, and Hpa I sites of the 152 kb Atriplex triangularis chloroplast genome, and the Sal I and Pvu II sites of the 155 kb Cucumis sativa chloroplast genome. These two chloroplast DNAs are very similar in organization; both feature the near-universal chloroplast DNA inverted repeat sequence of 22-25 kb. The positions of four different genes have been localized on these chloroplast DNAs. In both genomes the 16S and 23S ribosomal RNAs are encoded by duplicate genes situated at one end of the inverted repeat, while genes for the large subunit of ribulose-1,5-bisphosphate carboxylase and a 32 kilodalton photosystem II polypeptide are separated by 55 kb of DNA within the large single copy region. The physical and genetic organization of these DNAs is compared to that of spinach chloroplast DNA.     

Plastid-DNA levels in the different tissues of potato

The plastid-(pt) DNA levels in the different tissues of potato (Solanum tuberosum L.), including tubers of differing ages, have been studied. The DNA could be detected as a single nucleoid in amyloplasts of cells from young potato tubers by fluorescence microscopy, following staining of glutaraldehyde-fixed tissue with 4,6-diamidino-2-phenyl indole (DAPI). The renaturation kinetics of spinach ptDNA in the presence of total DNA from potato tissues and the fragments generated by restriction-enzyme digestion of potato-tuber DNA and chloroplast DNA indicated that the ptDNA of potato-tuber amyloplasts and of potato-leaf chloroplasts is essentially the same. Expressed as a percentage of the total DNA the level of ptDNA (5.2%) found in tubers, while less than that found in leaves (7.6%) was more than that found in petioles (3.4%), stems (3.0%) and roots (1.0%). There was a high level of both nuclear and plastid ploidy in mature potato-tuber cells and, on average, nuclei contained 32 pg of DNA (equivalent to 14C) and the 40 amyloplasts per cell contained DNA equivalent to 7800 copies of ptDNA, or 195 copies per amyloplast.Abbreviations DAPI 4,6-diamidino-2-phenyl-indole - LSU large sub-unit of ribulose-1,5-bisphosphate carboxylase - mtDNA mitochondrial DNA - ptDNA chloroplast or plastid DNA     

Nicotiana chloroplast genes for components of the photosynthetic apparatus

In order to understand more fully chloroplast genetic systems, we have determined the complete nucleotide sequence (155, 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA. It contains two copies of an identical 25,339 bp inverted repeat, which are separated by 86, 684 bp and 18,482 bp single-copy regions. The genes for 4 different rRNAs, 30 different tRNAs, 44 different proteins and 9 other predicted protein-coding genes have been located. Fifteen different genes contain introns.Twenty-two genes for components of the photosynthetic apparatus have so far been identified. Most of the genes (except the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase) code for thylakoid membrane proteins. Twenty of them are located in the large single-copy region and one gene for a 9-kd polypeptide of photosystem I is located in the small single-copy region. The gene for the 32-kd protein of photosystem II as well as the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase have strong promoters and are transcribed monocistronically while the other genes are transcribed polycistronically. We have found that the predicted amino acid sequences of six DNA sequences resemble those of components of the respiratory-chain NADH dehydrogenase from human mitochondria. As these six sequences are highly transcribed in tobacco chloroplasts, they are probably genes for components of a chloroplast NADH dehydrogenase. These observations suggest the existence of a respiratory-chain in the chloroplast of higher plants.     

The gene for the large subunit of ribulose-1,5-bisphosphate carboxylase in Euglena gracilis chloroplast DNA: location, polarity, cloning, and evidence for an intervening sequence

The gene for the large subunit (LS) of ribulose-1,5,-bisphosphate carboxylase of Euglena gracilis Z chloroplast DNA has been mapped by heterologous hybridization with DNA restriction fragments containing internal sequences from the Zea mays and Chlamydomonas reinhardii LS genes. The Euglena LS gene which has the same polarity as the Euglena rRNA genes has been located with respect to Pst I, Pvu I, and HindIII sites within the Eco RI fragment Eco A. The region of Euglena chloroplast DNA complementary to an 887 bp internal fragment from the Chlamydomonas chloroplast LS gene is interrupted by a 0.5-1.1 kbp non-complementary sequence. This is the first chloroplast protein gene located on the Euglena genome, and the first evidence for an intervening sequence within any chloroplast protein gene.     

Subunit polypeptide composition of rubisco and the origin of allopolyploid Arabidopsis suecica (Brassicaceae)

The polypeptide composition of the large and small subunits of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) from Arabidopsis thaliana, A. suecica and Cardaminopsis arenosa have been studied by IEF (isoelectric focusing) analysis. The putative recent alopolyploid origin of A. suecica is supported. The chloroplast encoded large subunits served to identify solely A. thaliana as the maternal parent whereas the nuclear encoded small subunits indicate C. arenosa as the paternal species.     

Functional loss of all ndh genes in an otherwise relatively unaltered plastid genome of the holoparasitic flowering plant Cuscuta reflexa

We have cloned and sequenced an area of about 9.0 kb of the plastid DNA (ptDNA) from the holoparasitic flowering plant Cuscuta reflexa to investigate the evolutionary response of plastid genes to a reduced selective pressure. The region contains genes for the 16S rRNA, a subunit of a plastid NAD(P)H dehydrogenase (ndhB), three transfer RNAs (trnA, trnI, trnV) as well as the gene coding for the ribosomal protein S7 (rps7). While the other genes are strongly conserved in C. reflexa, the ndhB gene is a pseudogene due to many frameshift mutations. In addition we used heterologous gene probes to identify the other ndh genes encoded by the plastid genome in higher plants. No hybridization signals could be obtained, suggesting that these genes are either lost or strongly altered in the ptDNA of C. reflexa. Together with evidence of deleted genes in the ptDNA of C. reflexa, the plastid genome can be grouped into four classes reflecting a different evolutionary rate in each case. The phylogenetic position of Cuscuta and the significance of ndh genes in the plastid genome of higher plants are discussed.     

香蕉rbcS基因启动子的克隆及序列分析

以巴西香蕉为材料,根据已经获得的香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的全长cDNA序列设计1对专一引物,通过PCR扩增得到了香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基的基因组全长,序列长811 bp,含有2个内含子。根据其基因组序列设计引物,采用SEFA-PCR方法,以总DNA为模板克隆了香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的启动子序列,长1 681 bp。用PLACE软件分析发现该序列具有启动子的基本元件TATA-box、CAAT-box,包含多个胁迫诱导元件,如光诱导元件、赤霉素、低温诱导元件、昼夜节律调控元件等。该序列的克隆与分析为进一步研究香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的表达调控奠定了基础。     

Cyanelle DNA from Cyanophora paradoxa

Summary Cyanelles which have been found in few eukaryotic organisms are photosynthetically active organelles which strikingly resemble cyanobacteria. The complexity of the cyanelle genome in Cyanophora paradoxa (127 Kbp) is too low to consider them as independent organisms in a symbiotic relationship. In order to correlate cyanelle genome and gene structure with those of plastid chromosomes of other plants, a circular map of the cyanelle DNA from Cyanophora paradoxa (strain LB555 UTEX) has been constructed using the restriction endonucleases SalI (generating 6 DNA fragments), BamHI (6), SalI (5), XhoI (9), and BglII (19).Besides the rRNA genes (16S, 23S, 5S), genes for 14 proteins have been located on this circular map. Among those are components of several multienzyme complexes involved in photosynthetic electron transport, as well as the large subunit of ribulose-1,5-bisphosphate carboxylase and two ribosomal proteins. All the probes used, were derived from a collection of spinach chloroplast DNA clones. Hybridization experiments showed signals to DNA fragments primarily from the large single-copy region of cyanelle DNA. The arrangement of genes on cyanelle DNA is different from that on spinach chloroplast DNA. However, genes which have been shown to be cotranscribed in spinach chloroplasts are also clustered on cyanelle DNA.Abbreviations Kbp 10³ base pairs - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase holoenzyme     

Positioning of protein-coding genes on the soybean chloroplast genome

Seven major plastid protein encoding genes were positioned on the soybean chloroplast DNA by heterologous hybridization. These include the genes for the alpha, beta and epsilon subunits of the CF₁ component of ATP synthase (atpA, atpB and atpE respectively), for subunit III of the CF₀ component of ATP synthase (atpH), for the cytochrome f (cytF), for the ‘32 Kd’ thylakoid protein (psbA), and for the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcL), all of which map in the large single copy region. The atpB, atpE and rbcL genes are located in the region adjacent to one of the segments of the inverted repeat. The genetic organization of the soybean chloroplast DNA is compared to that of other plastid genomes.     

Complementation of the nuclear antisense rbcS-induced photosynthesis deficiency by introducing an rbcS gene into the tobacco plastid genome

The small subunit (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a nuclear gene-encoded protein that is imported into chloroplasts where it assembles with the large subunit (LS) after removal of the transit peptide to form Rubisco. We have explored the possibility that the severe deficiency in photosynthesis exhibited in nuclear transgenic tobacco (line alpha5) expressing antisense rbcS coding DNA that results in low SS and Rubisco protein content [Rodermel et al. (1988) Cell 55: 673] could be complemented by introducing a copy of the rbcS gene into its plastid genome through chloroplast transformation. Two independent lines of transplastomic plants were generated, in which the tobacco rbcS coding sequence, either with or without the transit sequence, was site-specifically integrated into the plastid genome. We found that compared with the antisense plants, expression of the plastid rbcS gene in the transplastomic plants resulted in very high mRNA abundance but no increased accumulation of the SS and Rubisco protein or improvement in plant growth and photosynthesis. Therefore, there is a limitation in efficient translation of the rbcS mRNA in the plastid or an incorrect processing and modification of the plastid-synthesized SS protein that might cause its rapid degradation.     

Rice chloroplast DNA molecules are heterogeneous as revealed by DNA sequences of a cluster of genes.

We describe the isolation of two rice chloroplast HindIII fragments (9.5 kb and 5.3 kb) each containing a gene cluster coding for the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL), beta and epsilon subunits of ATPase (atpB and atpE), tRNAmet (trnM) and tRNAval (trnV). All five genes contained in the 9.5 kb fragment are potentially functional, whereas in the 5.3 kb fragment, rbcL is truncated and atpB is frame-shift mutated. The copy number of the 9.5 kb fragment is 10 times that of the 5.3 kb fragment, indicating that the two fragments are probably located on different chloroplast genomes and represent two different (major and minor) genomic populations. Thus, the rice chloroplast genome appears to be heterogeneous, contrary to general belief. We also describe the isolation of a rice mitochondrial HindIII fragment (6.9 kb) which contains an almost complete transferred copy of this chloroplast gene cluster. In this transferred copy, the coding sequences of rbcL, atpE and trnM contain perfectly normal reading frames, whereas atpB has become grossly defective and trnV is truncated.     

Rice chloroplast DNA: a physical map and the location of the genes for the large subunit of ribulose 1,5-bisphosphate carboxylase and the 32 KD photosystem II reaction center protein

Summary By homogenizing rice leaves in liquid nitrogen, it was possible to isolate intact chloroplasts and, subsequently, pure rice chloroplast DNA from the purified chloroplasts. The DNA was digested by several restriction enzymes and fragments were fractionated by agarose gel electrophoresis. The sum of the fragment sizes generated by the restriction enzymes showed that the total length of the DNA is 130 kb. A circular physical map of fragments, generated by digestion with SalI, PstI, and PvuII, has been constructed. The circular DNA contains two inverted repeats of about 20 kb separated by a large, single copy region of about 75 kb and a short, single copy region of about 15 kb. The location of the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase (Fraction I protein) and the 32 KD photosystem II reaction center gene were determined by using as probes tobacco chloroplast DNAs containing these genes. Rice chloroplast DNA differs from chloroplast DNAs of wheat and corn as well as from dicot chloroplast DNAs by having the 32 KD gene located 20 kb removed from the end of an inverted repeat instead of close to the end, as in other plants.     

Restriction endonuclease analysis of plastid DNA from tomato, potato and some of their somatic hybrids

Summary The buoyant density and endonuclease restriction patterns of potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum) ptDNA were examined and compared with those of their somatic hybrids. The plastids from these plants, both of which belong to the family of Solanaceae, contain a single DNA species whose density of 1.697 gcm^-3 and size of approximately 156 kbp are similar to those of ptDNA from other higher plants. The Sal I restriction patterns were indistinguishable; however, those obtained with Kpn I, Pst I, and Eco RI disclosed that each species possesses a unique ptDNA. These observations suggest a relatively recent divergence of both species. Of the twelve hybrid lines screened, eight contained exclusively potato ptDNa and four contained only tomato ptDNA at a 0.1–3% level of detection. Rearrangements of modifications of DNA were not detected. The plastid identities of three hybrid lines that had previously been analyzed by isoelectric focusing of RuBPcase subunits (Melchers et al. 1978) agreed with those determined by restriction endonuclease analysis.Abbreviations used in the text ptDNA plastid DNA, chloroplast DNA - cDNA copy DNA - RuBPcase ribulose bisphosphate carboxylase/oxygenase - LSU large subunit of RuBPcase - kbp kilobase pairs - SDS sodium dodecyl sulfate - SSC standard saline citrate - IEF isoelectric focusing