The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression.

The complete nucleotide sequence (155 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA has been determined. It contains two copies of an identical 25 339 bp inverted repeat, which are separated by a 86 684 bp and a 18 482 bp single-copy region. The genes for 4 different rRNAs, 30 different tRNAs, 39 different proteins and 11 other predicted protein coding genes have been located. Among them, 15 genes contain introns. Blot hybridization revealed that all rRNA and tRNA genes and 27 protein genes so far analysed are transcribed in the chloroplast and that primary transcripts of the split genes hitherto examined are spliced. Five sequences coding for proteins homologous to components of the respiratory-chain NADH dehydrogenase from human mitochondria have been found. The 30 tRNAs predicted from their genes are sufficient to read all codons if the ;two out of three' and ;U:N wobble' mechanisms operate in the chloroplast. Two sequences which autonomously replicate in yeast have also been mapped. The sequence and expression analyses indicate both prokaryotic and eukaryotic features of the chloroplast genes.

Synthesis and Assembly of the Polypeptides of Photosystem I and II in Isolated Etiochloroplasts of Wheat.

Synthesis and assembly of photosystems (PS) I and II polypeptides in etiochloroplasts isolated from greening wheat (Triticum aestivum L. cv Norin 61) seedlings were studied. The isolated etiochloroplasts synthesized PSI polypeptides of 66 and 15 kilodaltons, PSII polypeptides of 46 and 42 kilodaltons, and atrazine-binding 34 to 32 kilodalton polypeptide. Their assembly processes in the thylakoid membrane were studied by pulse-chase labeling with [(35)S]methionine, mild solubilization of the thylakoid membrane with Triton X-100, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis. The newly synthesized polypeptides of 66, 46, 42, 34, and 32 kilodaltons were first integrated into the complexes of 7.5, 5.9, 7.5, 6.3, and 7.5 Svedberg units, respectively, in 20 minutes. After the chase with excess amount of methionine for 100 min, they were found in complexes of 9.5, 9.1, 9.1, 9.1, and 9.1 Svedberg units, respectively. In this condition, stained polypeptides of PSI and PSII were found in the complexes of 11.1 and 10.3 Svedberg units, respectively. These results indicated that newly synthesized PSI or PSII polypeptides are integrated into intermediate complexes, but not complete complexes in the isolated etiochloroplasts. The relationship between the processing of the atrazine-binding 32 kilodalton polypeptide and its assembly into the PSII complex is also discussed.

Synthesis of a photosystem I polypeptide of 15 kilodaltons in isolated etiochloroplasts of wheat.

Etiochloroplasts isolated from greening wheat (Triticum aestivum L. cv Norin 61) seedlings synthesized a membrane polypeptide of 15 kilodaltons. One-dimensional peptide mapping with Staphylococcus aureus V8 protease revealed that the 15 kilodaltons polypeptide is the subunit 5 of photosystem I reaction center complex.

Organization of the psaH gene family of photosystem I in Nicotiana sylvestris.

The PSI-H subunit of photosystem I has two isoforms of differing molecular mass in Nicotiana sylvestris [Obokata et al. (1993) Plant Physiol. 102: 1259], and is encoded by a nuclear gene, psaH. We identified three structurally distinct psaH genes in the nuclear genome of N. sylvestris, designated psaHa, psaHb, and psaHc, and all three genes are expressed in young leaves. Each gene has two introns: one between sequences encoding a transit peptide and the N-terminal acidic domain, and one between the N-terminal domain and a central hydrophobic domain. The deduced amino acid sequences are identical in the mature proteins and differ only in the transit peptides. Since PSI-H is present in two isoforms in N. sylvestris, the psaH products may be subjected to post-translational modifications.

Tobacco nuclear genes for Photosystem I subunits, psaD, psaE and psaH share an octamer motif bound with nuclear proteins.

In Nicotiana sylvestris, nuclear-encoded photosystem I (PSI) genes, psaD, psaE and psaH, share an octamer motif bound with three phosphoproteins. This motif is not found in the chloroplast genome. From the view point of endosymbiont hypothesis, these results suggest that a set of ancient PSI genes acquired a common cis-element in the nucleus after they were transferred from the ancestral organelle.

Structure and expression of a nuclear gene for the PSI-D subunit of photosystem I in Nicotiana sylvestris.

The PSI-D subunit is the ferredoxin-binding site of photosystem I, and is encoded by the nuclear gene psaD. We isolated a psaD genomic clone from Nicotiana sylvestris, by screening a genomic library with a psaD cDNA which we previously cloned from N. sylvestris (Yamamoto et al., Plant Mol Biol 17: 1251, 1991). Nucleotide sequence analysis revealed that this genomic clone contains a psaD gene, which does not correspond to the psaD cDNA, so we designated these genes psaDb and psaDa, respectively. The psaDb clone encodes a protein of 214 amino acids uninterrupted by introns. The N-terminal sequence determined for the N. sylvestris PSI-D protein encoded by psaDb begins at the 49th residue. The products of psaDa and psaDb share 82.7% and 79.5% identity at the amino acid and nucleotide levels, respectively. Genomic Southern analysis showed that two copies of psaD are present in the N. sylvestris genome. Ribonuclease protection assays and immunoblot analysis in N. sylvestris indicate that both genes are expressed in leaves, stems and flower buds, but neither is expressed in roots. During leaf development, the ratio of psaDb to psaDa mRNA increases from 0.12 in leaf buds to 0.36 in mature leaves. The relative abundance of the corresponding proteins decreased over the same developmental period. These results indicate that differential regulation mechanisms control psaDa and psaDb expression at both the mRNA and protein levels during leaf development.

5'-leader of a photosystem I gene in Nicotiana sylvestris, psaDb, contains a translational enhancer.

Messenger RNA primary structures responsible for translational efficiency of a photosystem I gene, psaDb, of Nicotiana sylvestris were studied using a transgenic tobacco system. The entire 5'-leader (23 base pairs) with the first four amino acid codons of the protein coding region was fused in frame with the beta-glucuronidase (GUS) gene under the control of the 35 S promoter of cauliflower mosaic virus (CaMV). This construct (CaMV::psaDb-GUS') was introduced into tobacco. GUS activity and GUS mRNA levels were determined for individual transformants, revealing that the insertion of the psaDb sequence greatly enhanced the GUS activity relative to GUS mRNA abundance. The GUS activity/GUS mRNA was 14 times higher in the CaMV::psaDb-GUS' transformants than in the control CaMV::GUS' transformants. The high GUS activity/GUS mRNA of the CaMV::psaDb-GUS' transformants was reduced 20-fold when 13 bases within the psaDb leader were altered. These 13 bases are common to the leaders of an Arabidopsis ferredoxin gene and the psaDb gene of N. sylvestris. Since GUS proteins encoded by these chimeric GUS genes have identical amino acid sequences, these results indicate that the 5'-leader of the psaDb mRNA contains a translational enhancer element.

Microheterogeneity of PSI-E subunit of photosystem I in Nicotiana sylvestris.

Microheterogeneity of a photosystem I (PSI) subunit encoded by a nuclear gene psaE was examined in Nicotiana sylvestris, with the aid of cDNA cloning, peptide mapping analysis and protein sequencing. The psaE product of this plant has four isoforms whose mobilities in PAGE are slightly different from each other. We isolated two types of psaE cDNAs from a N. sylvestris cDNA library, and designated the corresponding genes as psaEa and psaEb, respectively. The psaEa and psaEb genes are 77% homologous at DNA level, and their translation products share 80.4% homology for the precursor proteins and 89.1% for the mature forms. Comparative analysis of the four isoproteins and the putative products of the two psaE genes revealed that two isoproteins out of four are derived from psaEa gene, and the difference between these two isoproteins lies in the respective presence or absence of N-terminal alanine. Likewise, the other two proteins are derived from psaEb with similar N-terminal heterogeneity. These results indicate that multi-gene organization and heterogeneous N-terminal formation at post-translational level are two possible causes for PSI subunit polymorphism in isogenic plant lines.

Polymorphism of a photosystem I subunit caused by alloploidy in Nicotiana.

The photosystem I complex from Nicotiana tabacum, which has an alloploid genome, contains subunits of 17.5 and 18.5 kilodaltons whose N-terminal amino acid sequences are highly homologous. Comparative analysis of photosystem I subunits among N. tabacum and its ancestral plants, N. tomentosiformis and N. sylvestris, revealed that the 17.5 kilodalton subunit of N. tabacum derives from N. sylvestris, and the 18.5 kilodalton subunit from N. tomentosiformis.

Rapid splicing and stepwise processing of a transcript from the psbB operon in tobacco chloroplasts: determination of the intron sites in petB and petD.

Expression of the psbB gene cluster in tobacco chloroplasts has been studied. This cluster contains the genes for the 51 kDa chlorophyll a apoprotein (psbB) and the 10 kDa phosphoprotein (psbH) of the photosystem II, and cytochrome b6 (petB) and subunit IV (petD) of the cytochrome b/f complex in this order. Northern blot hybridization and reverse transcription analyses have revealed that petB and petD contain single introns and the psbB gene cluster is transcribed as a single polycistronic unit. The primary transcript seems to be spliced very rapidly and then processed into several small RNA species. The exact splice sites have been located by cDNA sequencing. The transcriptional initiation site of the psbB operon has been determined by S1 mapping with in vitro capped chloroplast RNA. The stepwise processing of chloroplast RNA precursors is discussed.

Molecular heterogeneity of photosystem I. psaD, psaE, psaF, psaH, and psaL are all present in isoforms in Nicotiana spp.

The protein composition of photosystem I (PSI) was examined in Nicotiana spp. by high-resolution polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and immunoblot analysis. Five PSI proteins show polymorphism in an amphidiploid species, Nicotiana tabacum, but not in its ancestral diploid species, Nicotiana sylvestris and Nicotiana tomentosiformis. These Nicotiana spp. appear to have at least 18 PSI proteins per genome that range in molecular mass from 3 to 20 kD. They include the products of nuclear genes psaD, psaE, psaF, psaG, psaH, psaK, and psaL, the product of chloroplast gene psaC, N-terminally blocked proteins of 4.5 and 3.0 kD, and an unidentified protein of 12.5 kD. The psaD, psaF, psaH, and psaL products have two isoforms each that are distinguished by different mobilities in polyacrylamide gel electrophoresis, and the psaE product has four isoforms. The two isoforms of the psaD product have distinct amino acid sequences, indicating that they are encoded by different genes within the genome. Four isoforms of the psaE products can be classified into two groups by N-terminal amino acid sequence, indicating that at least two psaE genes are present in the genome. To examine whether the polymorphic nature of PSI is peculiar to Nicotiana spp., we carried out immunoblot analysis of the psaD and psaE products in isogenic lines of tomato (Lycopersicon esculentum), Arabidopsis thaliana, red bean (Vigna angularis), and corn (Zea mays). Two electrophoretically distinct isoforms were found for the psaD products of tomato, A. thaliana, and corn, and two isoforms of psaE products were detected in tomato, A. thaliana, and red bean. These results suggest that the nuclear-encoded subunits of PSI, except for the psaG and psaK products, generally have two isoforms.

Light-responsive elements of the tobacco PSI-D gene are located both upstream and within the transcribed region.

psaDb is a nuclear gene encoding the ferredoxin-binding subunit of photosystem I in Nicotiana sylvestris. The organization of the light-responsive cis-elements of psaDb was studied using transgenic tobacco plants. Three types of psaDb chimeric constructs were created: (1) a 5' upstream fragment of psaDb transcriptionally fused with the beta-glucuronidase (GUS) gene, and a series of its 5' deletion derivatives, (2) the transcribed region of psaDb driven by the cauliflower mosaic virus (CaMV) 35S promoter, and (3) the 5' terminal 35 bases (the entire leader, +1 to +23, and the initiation codon context, +24 to +35) of the psaDb mRNA translationally fused with a GUS reporter gene under the operation of the CaMV 35S promoter. Light-responsiveness of these fusions in transgenic plants was examined by GUS assay and primer extension analysis. The results indicate that the light-responsive elements (LRE) of psaDb are located both upstream (-170 to +24) and within (+1 to +861) the transcribed region. The internal LRE is utilized in etiolated seedings but not in green leaves. The leader and initiation codon context construct (+1 to +35) did not show any light-response under the conditions tested. Therefore, it is likely that a combination of the upstream and internal LREs generates the complex light-responsive and tissue-specific regulation of this gene. This study also revealed that psaDb has adjacent activator (-267 to -254) and repressor (-253 to -234) regions for basal transcriptional activity; the former contains the ACGT binding motif recognized by many plant bZIP proteins, and the latter has the R3 decamer motif found in several photosystem I-related genes.