Organization and expression of the nuclear gene coding for the plastid-specific S22 ribosomal protein from spinach.

We report here on the genomic organization and expression of a nuclear gene coding for a plastid ribosomal protein. The gene encodes the plastid-specific ribosomal protein S22 (formerly named CS-S5). Southern blot analysis suggests that the gene is present in one copy in the spinach genome. The gene consists of 5 exons of sizes ranging from 108 to 273 bp and of 4 introns of 1410, 92, 386 and 82 bp. The exon-intron splice junctions and intron branch sites fit well the consensus sequences for plant introns. The major transcription start site has been determined 29 bp upstream of the AUG initiation codon by primer extension and S1 nuclease mapping. No canonical TATA box is found but some other possible promoter motifs are observed. Transcripts are detected in leaves, etiolated leaves, roots and seeds suggesting that the rps22 gene is expressed constitutively. During germination a marked increase in the relative steady-state level of the mRNA can be seen as soon as 24 h after imbibition of the seeds.

Molecular cloning of a small DNA binding protein with specificity for a tissue-specific negative element within the rps1 promoter.

A cDNA encoding a specific binding activity for the tissue-specific negative cis-element S1F binding site of spinach rps1 was isolated from a spinach cDNA expression library. This cDNA of 0.7 kb encodes an unusual small peptide of only 70 amino acids, with a basic domain which contains a nuclear localization signal and a putative DNA binding helix. This protein, named S1Fa, is highly conserved between dicotyledonous and monocotyledonous plants and may represent a novel class of DNA binding proteins. The corresponding mRNA is accumulated more in roots and in etiolated seedlings than in green leaves. This expression pattern is correlated with the tissue-specific function of the S1F binding site which represses the rps1 promoter preferentially in roots and in etiolated plants.

Mitochondrial rps14 is a transcribed and edited pseudogene in Arabidopsis thaliana.

We have isolated and analysed a 2 kb region of the mitochondrial genome of Arabidopsis thaliana (Columbia) showing a high level of nucleotide identity with the mitochondrial (mt) rps14 small-subunit ribosomal protein gene from Oenothera berteriana and Vicia faba, as well as with an open reading frame (ORF) located upstream of the nad3 locus in O. berteriana. The rps14 locus is present as a single copy in the A. thaliana mt genome and has a translational stop codon located near the initiation codon, as well as a deletion of one nucleotide that disturbs the coding sequence. The cloning and sequencing of nine amplified mt rps14 cDNAs clearly demonstrated that this gene is transcribed and that the mRNA precursors are edited at three positions, all involving C-to-U conversions. No editing events changing the stop codon and restoring the correct coding sequence were witnessed within the 9 individual cDNA clones. Therefore, we conclude that the single rps14 sequence of the mitochondrial genome from A. thaliana is in fact a pseudogene that is transcribed and edited but not translated.

The components of the plastid ribosome are not accumulated synchronously during the early development of spinach plants.

The expression of components of the 70S plastid ribosome has been determined during the first 13 days of spinach plant development. Total cellular RNA and proteins were used to determine the relative steady-state levels of mRNA for ribosomal proteins (r-proteins) by dot blot hybridization and the relative amounts of proteins by immunodetection with specific antibodies. The 16S rRNA as well as mRNAs for 9 out of 11 proteins studied, including those for the 32 kDa polypeptide of photosystem II and the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase (Rubisco) show a marked increase at the beginning of the germination (day 5). At this time the plastid DNA content increases from 4% to 6% of total DNA content and so the plastome copy number can only in part account for the important increase in mRNA steady-state levels. Interestingly the transcripts of the rpl23 and rps19 genes show a different accumulation pattern, indicating either a differential gene transcription and/or an increased stability of the transcripts. In the western blot analysis a group of r-proteins can be detected in dry seeds or after 24 hours of imbibition while a second group of proteins accumulates after 3 to 5 days of development. The differential accumulation pattern of r-proteins and mRNA for r-proteins indicates that post-transcriptional control plays an important role in plastid r-protein synthesis.

Structure and transcription of the 5S rRNA gene from spinach chloroplasts.

The nucleotide sequence of the spinach chloroplast 5S rRNA gene and its flanking regions has been determined. A prokaryotic type promoter is to be found upstream of the 5S rRNA gene. Northern blot experiments with selected gene probes show that the 5S gene is co-transcribed with the other ribosomal genes of the operon. This result is confirmed by 5' S1 mapping of in vivo RNAs synthesised in chloroplasts or in an E. coli strain harboring a multicopy plasmid containing the 5S rRNA gene and its flanking regions. In vitro transcription experiments show that initiation of transcription does not occur at the level of the putative 5S rRNA gene promoter. Therefore, we conclude that the 5S rRNA is synthesized only be co-transcription of its gene with the other ribosomal genes of the operon. 3' S1 nuclease mapping in the spacer region between the 4.5S and the 5S rRNA genes reveals a set of protected fragments located in an A.T rich region downstream of a very stable hairpin and immediately upstream of the putative 5S promoter. This result is interpreted by the presence of preterminated transcripts or processing sites in this region.

In vitro transcription initiation of the spinach chloroplast 16S rRNA gene at two tandem promoters.

Two potential prokaryotic promoters, P1 and P2, are characterized 164 and 114 bp upstream of the spinach chloroplast 16S rRNA gene. The strengths of these promoters, calculated according to an homology score established for E. coli RNA-polymerase, are identical. Experiments performed with a Taq I-DNA fragment, containing 16 bp of the 16S rDNA and 243 bp upstream of the gene, give evidence that in vitro, E. coli RNA-polymerase starts transcription at these two promoters. These results are based on both the size of the transcripts and their nucleotide sequences. A possible regulation by differential control of these dual promoters is suggested. S1 mapping with RNAs extracted either from green or from etiolated spinach plants, indicates that, at these two steps of plastid development, transcription in vivo starts at P1. Surprisingly only P2 appears to be conserved in the homologous sequences reported for maize, mustard and Spirodela.

The expression of nuclear genes encoding plastid ribosomal proteins precedes the expression of chloroplast genes during early phases of chloroplast development.

The development of different plant organs (root, hypocotyl, and cotyledons) during seed germination is connected with the transformation of proplastids, which are found in embryonic and meristematic tissues, into amyloplasts in root tissues and into chloroplasts in cotyledons. We have analyzed the expression of nuclear and plastid genes coding for the plastid translational apparatus during the first 7 d of Spinacia oleracea development. Results show that the nuclear genes (rps1, rps22, rpI21, and rpI40) are expressed from the 1st d of seed imbibition and precede transcription of the chloroplast-encoded genes (photosynthetic and nonphotosynthetic), which starts the 3rd d after the beginning of imbibition. Transcription from the leaf-/cotyledon-specific P1 promoter of the rpI21 gene starts on the first imbibition day. Inhibition of chloroplast biogenesis by bleaching in the presence of norflurazon has no influence on the expression from this P1 promoter, suggesting that the onset of transcription of nuclear gene rpI21 is independent of a plastid signal.

Cis-acting elements and expression pattern of the spinach rps22 gene coding for a plastid-specific ribosomal protein.

In order to study the regulation of nuclear genes coding for plastid ribosomal proteins, we have analysed the promoter region of spinach rps22 using both in vitro and in vivo approaches. By footprinting analyses, we have identified eight DNA elements interacting with spinach leaf nuclear factors in the 300 bp promoter region upstream of the transcription start site. Among these elements, four are short AT-rich sequences and one is identical to the Hex motif characterized initially in wheat histone genes. In transgenic tobacco plants, the reporter gene coding for the beta-glucuronidase (GUS) directed by a 1.2 kb upstream region of rps22 was expressed in several plant organs, with high levels in leaf mesophyll, embryo cotyledons and root meristematic cells and very low levels in other cell types. Interestingly, when deleted to -295, the promoter, which contained all the foot-printed elements, was still able to confer the same expression pattern, although the activity was relatively lower than with the 1.2 kb promoter. When deleted further to -154, the promoter, from which the AT-rich elements were eliminated, loses its activity almost completely, suggesting that these AT-rich elements are important for the rps22 promoter activity. Altogether, our results show that rps22 gene expression is controlled by specific cis elements not present in other nuclear-encoded plastid ribosomal protein genes studied so far.

Hypothesis for the evolutionary origin of the chloroplast ribosomal protein L21 of spinach.

A full size cDNA clone encoding the chloroplast ribosomal protein L21 from spinach is presented. The identity of the clone and the location of the transit peptide processing site were determined by comparison with the N-terminal amino acid sequence of the spinach chloroplast protein CS-L7 previously identified. L21 r-protein sequences from spinach, Marchantia polymorpha and Escherichia coli are compared. Quite surprisingly, the data do not suggest that the rpl21 nuclear gene from spinach was derived through intracellular gene transfer from the chloroplast genome. The possibility of a mitochondrial origin for rpl21 gene of spinach is discussed.