Sequence-specific interaction between S1F, a spinach nuclear factor, and a negative cis-element conserved in plastid-related genes.

The nuclear gene rps1 coding for the spinach plastid ribosomal protein CS1 exhibits both a constitutive and leaf-specific expression pattern. In contrast to other chloroplast-related genes like rbcS and cab, the leaf induction of rps1 expression is light-independent. These unique features of rps1 expression provide good models to study the mechanisms regulating plastid development and differentiation in higher plants. We report on the identification of a spinach leaf nuclear factor, designated S1F, interacting with the rps1 promoter. The S1F binding site is conserved in the promoter region of many plastid-related genes, including rbcS, cab, and rpl21. A binding activity similar to S1F was detected in nuclear extract from dark-grown de-differentiated soybean suspension cells. Through site-specific mutagenesis and transient expression in soybean cell protoplasts, we show that the S1F binding site is a negative element down-regulating the promoter activity of rps1. A ligated tetramer of S1F site was able to repress activity of the cauliflower mosaic virus 35 S promoter extending the negative function of the S1F binding site on promoter activity.     

Complete plastid genome sequence of the chickpea (Cicer arietinum) and the phylogenetic distribution of rps12 and clpP intron losses among legumes (Leguminosae)

Chickpea (Cicerarietinum, Leguminosae), an important grain legume, is widely used for food and fodder throughout the world. We sequenced the complete plastid genome of chickpea, which is 125,319bp in size, and contains only one copy of the inverted repeat (IR). The genome encodes 108 genes, including 4 rRNAs, 29 tRNAs, and 75 proteins. The genes rps16, infA, and ycf4 are absent in the chickpea plastid genome, and ndhB has an internal stop codon in the 5'exon, similar to other legumes. Two genes have lost their introns, one in the 3'exon of the transpliced gene rps12, and the one between exons 1 and 2 of clpP; this represents the first documented case of the loss of introns from both of these genes in the same plastid genome. An extensive phylogenetic survey of these intron losses was performed on 302 taxa across legumes and the related family Polygalaceae. The clpP intron has been lost exclusively in taxa from the temperate "IR-lacking clade" (IRLC), whereas the rps12 intron has been lost in most members of the IRLC (with the exception of Wisteria, Callerya, Afgekia, and certain species of Millettia, which represent the earliest diverging lineages of this clade), and in the tribe Desmodieae, which is closely related to the tribes Phaseoleae and Psoraleeae. Data provided here suggest that the loss of the rps12 intron occurred after the loss of the IR. The two new genomic changes identified in the present study provide additional support of the monophyly of the IR-loss clade, and resolution of the pattern of the earliest-branching lineages in this clade. The availability of the complete chickpea plastid genome sequence also provides valuable information on intergenic spacer regions among legumes and endogenous regulatory sequences for plastid genetic engineering.     

Junctions of the large single copy region and the inverted repeats in Spinacia oleracea and Nicotiana debneyi chloroplast DNA: sequence of the genes for tRNAHis and the ribosomal proteins S19 and L2.

This work describes the organization, at the nucleotide sequence level, of genes flanking the junctions of the large single copy regions and the inverted repeats of Spinacia oleracea (spinach) and Nicotiana debneyi chloroplast DNAs. In both genomes, trnH1, the gene for tRNA-His(GUG) is located at the extremity of the large single copy region 3' to psbA, the gene for the 35 kd Photosystem 2 protein. Both psbA and trnH1 are transcribed towards the inverted repeat. In spinach, the first 48 codons of rps19, the gene for the chloroplast ribosomal protein S19, lie in the inverted repeat and the last 44 codons lie in the large single copy region at the end opposite to that carrying trnH1. The gene for a protein homologous to the E. coli ribosomal protein L2, rp12, is in the inverted repeat immediately 5' to rps19 and, like rps19, is transcribed towards the large single copy region. In N. debneyi, but not in spinach, rp12 is interrupted by a 666 bp insertion. The gene for tRNA-lle(CAT), trnl1, is located in the inverted repeats of spinach and N. debneyi, 5' to rp12 and is transcribed in the same direction as rp12.     

Do nonasterid holoparasitic flowering plants have plastid genomes?

Past work involving the plastid genome (plastome) of holoparasitic plants has been confined to Scrophulariaceae (or Orobanchaceae) which have truncated plastomes owing to loss of photosynthetic and other genes. Nonasterid holoparasites from Balanophoraceae (Corynaea), Hydnoraceae (Hydnora) and Cytinaceae (Cytinus) were tested for the presence of plastid genes and a plastome. Using PCR, plastid 16S rDNA was successfully amplified and sequenced from the above three holoparasites. The sequence of Cytinus showed 121 single base substitutions relative to Nicotiana (8% of the molecule) whereas higher sequence divergence was observed in Hydnora and Corynaea (287 and 513 changes, respectively). Secondary structural models for these 16S rRNAs show that most changes are compensatory, thus suggesting they are functional. Probes constructed for 16S rDNA and for four plastid-encoded ribosomal protein genes (rps2, rps4, rps7 and rpl16) were used in Southern blots of digested genomic DNA from the three holoparasites. Positive hybridizations were obtained using each of the five probes only for Cytinus. For SmaI digests, all plastid gene probes hybridized to a common fragment ca. 20 kb in length in this species. Taken together, these data provide preliminary evidence suggestive of the retention of highly diverged and truncated plastid genome in Cytinus. The greater sequence divergence for 16S rDNA and the negative hybridization results for Hydnora and Corynaea suggests two possibilities: the loss of typically conserved elements of their plastomes or the complete absence of a plastome.     

A first molecular phylogenetic analysis of Passiflora (Passifloraceae)

Passiflora, a genus with more than 400 species, exhibits a high diversity of floral and vegetative structures and a complex taxonomy, which includes 23 subgenera and many sections and series. To better understand Passiflora's variability and interspecific relationships, the phylogeny of 61 species, classified in 11 of 23 suggested subgenera, was investigated. Three molecular markers were used, the nuclear ribosomal internal transcribed spacers (nrITS), the plastid trnL-trnF spacer regions (～1000 bp), and the rps4 plastid gene (～570 bp). Three major clades were highly supported, independent of the marker and phylogenetic method used; one included the subgenera Distephana, Dysosmia, Dysosmioides, Passiflora, and Tacsonioides, a second, the subgenera Adopogyne, Decaloba, Murucuja, and Pseudomurucuja, and a third, the subgenus Astrophea. We call these the Passiflora, Decaloba, and Astrophea clades, respectively. The position of subgenus Deidamioides is undefined. The monophyly of Passiflora could not be statistically corroborated, and the relationships among the major clades and of these clades with the related genera remain unresolved. Our results indicate that a reevaluation of the monophyly of Passiflora and its infrageneric classification is necessary.     

Tobacco plastid ribosomal protein S18 is essential for cell survival

Plastid genomes contain a conserved set of genes most of which are involved in either photosynthesis or gene expression. Among the ribosomal protein genes present in higher plant plastid genomes, rps18 is special in that it is absent from the plastid genomes of several non-green unicellular organisms, including Euglena longa and Toxoplasma gondii. Here we have tested whether the ribosomal protein S18 is required for translation by deleting the rps18 gene from the tobacco plastid genome. We report that, while deletion of the rps18 gene was readily obtained, no homoplasmic Δrps18 plants or leaf sectors could be isolated. Instead, segregation into homoplasmy led to severe defects in leaf development suggesting that the knockout of rps18 is lethal and the S18 protein is required for cell survival. Our data demonstrate that S18 is indispensable for plastid ribosome function in tobacco and support an essential role for plastid translation in plant development. Moreover, we demonstrate the occurrence of flip-flop recombination on short inverted repeat sequences which generates different isoforms of the transformed plastid genome that differ in the orientation a 70 kb segment in the large single-copy region. However, infrequent occurrence of flip-flop recombination and random segregation of plastid genomes result in the predominant presence of only one of the isoforms in many tissue samples. Implications for the interpretation of chloroplast transformation experiments and vector design are discussed.     

Nonessential plastid-encoded ribosomal proteins in tobacco: a developmental role for plastid translation and implications for reductive genome evolution

Plastid genomes of higher plants contain a conserved set of ribosomal protein genes. Although plastid translational activity is essential for cell survival in tobacco (Nicotiana tabacum), individual plastid ribosomal proteins can be nonessential. Candidates for nonessential plastid ribosomal proteins are ribosomal proteins identified as nonessential in bacteria and those whose genes were lost from the highly reduced plastid genomes of nonphotosynthetic plastid-bearing lineages (parasitic plants, apicomplexan protozoa). Here we report the reverse genetic analysis of seven plastid-encoded ribosomal proteins that meet these criteria. We have introduced knockout alleles for the corresponding genes into the tobacco plastid genome. Five of the targeted genes (ribosomal protein of the large subunit22 [rpl22], rpl23, rpl32, ribosomal protein of the small subunit3 [rps3], and rps16) were shown to be essential even under heterotrophic conditions, despite their loss in at least some parasitic plastid-bearing lineages. This suggests that nonphotosynthetic plastids show elevated rates of gene transfer to the nuclear genome. Knockout of two ribosomal protein genes, rps15 and rpl36, yielded homoplasmic transplastomic mutants, thus indicating nonessentiality. Whereas Δrps15 plants showed only a mild phenotype, Δrpl36 plants were severely impaired in photosynthesis and growth and, moreover, displayed greatly altered leaf morphology. This finding provides strong genetic evidence that chloroplast translational activity influences leaf development, presumably via a retrograde signaling pathway.     

Inferring the history of the polyploid Silene aegaea (Caryophyllaceae) using plastid and homoeologous nuclear DNA sequences

The origin of the rare allotetraploid Silene aegaea was inferred from plastid rps16 intron sequences, homoeologous copies of nuclear ribosomal internal transcribed spacer (ITS) sequences, and an intron from the nuclear gene coding for the second largest subunit of RNA polymerase II (RPB2). The nuclear DNA regions support the S. sedoides and S. pentelica lineages as most closely related to the two S. aegaea paralogues. A few recombinant ITS sequences were found, but as PCR recombination could be demonstrated, no true recombination could be demonstrated. No recombination was found in the RPB2 sequences. Plastid rps16 intron sequences strongly support S. pentelica as the maternal lineage. The strength of the approach of using homoeologous sequences of several loci is demonstrated, and its usefulness for the study of phylogenies of groups including polyploids is emphasized.