Loss of the rpl32 gene from the chloroplast genome and subsequent acquisition of a preexisting transit peptide within the nuclear gene in Populus

Gene transfer events from organelle genomes (mitochondria and chloroplasts in plants) to the nuclear genome are important processes in the evolution of the eukaryotic cell. It is highly likely that the gene transfer event is still an ongoing process in higher plant mitochondria and chloroplasts. The number and order of genes encoded in the chloroplast genome of higher plants are highly conserved. Recently, several exceptional cases of gene loss from the chloroplast genome have been discovered as the number of complete chloroplast genome sequences has increased. The Populus chloroplast genome has lost the rpl32 gene, while the corresponding the chloroplast rpl32 (cp rpl32) gene has been identified in the nuclear genome. Nuclear genes transferred from the chloroplast genome need to gain a sequence that encodes a transit peptide. Here, we revealed that the nuclear cp rpl32 gene has acquired the exon sequence, which is highly homologous to a transit peptide derived from the chloroplast Cu-Zn superoxide dismutase (cp sod-1) gene. The cp rpl32 gene has acquired the sequence that encodes not only for the transit peptide, but also for the conserved N-terminal portion of the mature SOD protein from the cp sod-1 gene, suggesting the occurrence of DNA sequence duplication. Unlike cp SOD-1, cp RPL32 did not show biased localization in the chloroplasts. This difference may be caused by mutations accumulated in the sequence of the SOD domain on the cp rpl32 gene. We provide new insight into the fate of the inherent sequence derived from a transit peptide.     

Characterization of two rice genes for nuclear-encoded chloroplast ribosomal protein L12 and phylogenetic analysis of the acquisition of transit peptides and gene duplication

 We have identified two genes coding for chloroplast ribosomal protein L12 encoded in the nuclear genome of rice (Oryza sativa). These genes were designated rpl12-1 and rpl12-2 (rpl12, ribosomal protein L12). Northern analysis with specific probes revealed that both genes are transcribed. The expression of each gene seems to have a different regulatory machinery. It is also suggested that the expression of rpl12-1 is controlled in an organ-specific manner. The deduced amino-acid sequences of the mature peptide parts are more conserved than those of the transit peptide parts in both monocotyledonous and dicotyledonous plants. A phylogenetic tree was constructed using the nucleotide sequences of the transit peptide region of the rpl12s of reported plant. The tree includes estimates of when the transit peptides were acquired, and when the genes were duplicated, in the course of evolution. According to our hypothesis, the nuclear-translocated chloroplast ribosomal protein L12 gene obtained its transit peptide after the divergence of monocots and dicots, then gene duplications occurred independently in monocots and dicots, and subsequently rice and rye branched apart. Received: 4 October 1997 / Accepted: 5 January 1998     

Many parallel losses of infA from chloroplast DNA during angiosperm evolution with multiple independent transfers to the nucleus

We used DNA sequencing and gel blot surveys to assess the integrity of the chloroplast gene infA, which codes for translation initiation factor 1, in >300 diverse angiosperms. Whereas most angiosperms appear to contain an intact chloroplast infA gene, the gene has repeatedly become defunct in approximately 24 separate lineages of angiosperms, including almost all rosid species. In four species in which chloroplast infA is defunct, transferred and expressed copies of the gene were found in the nucleus, complete with putative chloroplast transit peptide sequences. The transit peptide sequences of the nuclear infA genes from soybean and Arabidopsis were shown to be functional by their ability to target green fluorescent protein to chloroplasts in vivo. Phylogenetic analysis of infA sequences and assessment of transit peptide homology indicate that the four nuclear infA genes are probably derived from four independent gene transfers from chloroplast to nuclear DNA during angiosperm evolution. Considering this and the many separate losses of infA from chloroplast DNA, the gene has probably been transferred many more times, making infA by far the most mobile chloroplast gene known in plants.     

Complete plastid genome sequences of three Rosids (Castanea, Prunus, Theobroma): evidence for at least two independent transfers of rpl22 to the nucleus

Functional gene transfer from the plastid to the nucleus is rare among land plants despite evidence that DNA transfer to the nucleus is relatively frequent. During the course of sequencing plastid genomes from representative species from three rosid genera (Castanea, Prunus, Theobroma) and ongoing projects focusing on the Fagaceae and Passifloraceae, we identified putative losses of rpl22 in these two angiosperm families. We further characterized rpl22 from three species of Passiflora and one species of Quercus and identified sequences that likely represent pseudogenes. In Castanea and Quercus, both members of the Fagaceae, we identified a nuclear copy of rpl22, which consisted of two exons separated by an intron. Exon 1 encodes a transit peptide that likely targets the protein product back to the plastid and exon 2 encodes rpl22. We performed phylogenetic analyses of 97 taxa, including 93 angiosperms and four gymnosperm outgroups using alignments of 81 plastid genes to examine the phylogenetic distribution of rpl22 loss and transfer to the nucleus. Our results indicate that within rosids there have been independent transfers of rpl22 to the nucleus in Fabaceae and Fagaceae and a putative third transfer in Passiflora. The high level of sequence divergence between the transit peptides in Fabaceae and Fagaceae strongly suggest that these represent independent transfers. Furthermore, Blast searches did not identify the "donor" genes of the transit peptides, suggesting a de novo origin. We also performed phylogenetic analyses of rpl22 for 87 angiosperms and four gymnosperms, including nuclear-encoded copies for five species of Fabaceae and Fagaceae. The resulting trees indicated that the transfer of rpl22 to the nucleus does not predate the origin of angiosperms as suggested in an earlier study. Using previously published angiosperm divergence time estimates, we suggest that these transfers occurred approximately 56-58, 34-37, and 26-27 Ma for the Fabaceae, Fagaceae, and Passifloraceae, respectively.     

Complete structure of the chloroplast genome of a legume, Lotus japonicus.

The nucleotide sequence of the entire chloroplast genome (150,519 bp) of a legume, Lotus japonicus, has been determined. The circular double-stranded DNA contains a pair of inverted repeats of 25,156 bp which are separated by a small and a large single copy region of 18,271 bp and 81,936 bp, respectively. A total of 84 predicted protein-coding genes including 7 genes duplicated in the inverted repeat regions, 4 ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acids species were assigned on the genome based on similarity to genes previously identified in other chloroplasts. All the predicted genes were conserved among dicot plants except that rpl22, a gene encoding chloroplast ribosomal protein CL22, was missing in L. japonicus. Inversion of a 51-kb segment spanning rbcL to rpsl6 (positions 5161-56,176) in the large single copy region was observed in the chloroplast genome of L. japonicus. The sequence data and gene information are available on our World Wide Web database at http://www.kazusa.or.jp/en/plant/database.html.     

Genes for two mitochondrial ribosomal proteins in flowering plants are derived from their chloroplast or cytosolic counterparts

Often during flowering plant evolution, ribosomal protein genes have been lost from the mitochondrion and transferred to the nucleus. Here, we show that substitution by a duplicated, divergent gene originally encoding the chloroplast or cytosolic ribosomal protein counterpart accounts for two missing mitochondrial genes in diverse angiosperms. The rps13 gene is missing from the mitochondrial genome of many rosids, and a transferred copy of this gene is not evident in the nucleus of Arabidopsis, soybean, or cotton. Instead, these rosids contain a divergent nuclear copy of an rps13 gene of chloroplast origin. The product of this gene from all three rosids was shown to be imported into isolated mitochondria but not into chloroplasts. The rps8 gene is missing from the mitochondrion and nucleus of all angiosperms examined. A divergent copy of the gene encoding its cytosolic counterpart (rps15A) was identified in the nucleus of four angiosperms and one gymnosperm. The product of this gene from Arabidopsis and tomato was imported successfully into mitochondria. We infer that rps13 was lost from the mitochondrial genome and substituted by a duplicated nuclear gene of chloroplast origin early in rosid evolution, whereas rps8 loss and substitution by a gene of nuclear/cytosolic origin occurred much earlier, in a common ancestor of angiosperms and gymnosperms.     

A ribosomal protein is encoded in the chloroplast DNA in a lower plant but in the nucleus in angiosperms. Isolation of the spinach L21 protein and cDNA clone with transit and an unusual repeat sequence

The distribution of chloroplast ribosomal protein genes between the organelle DNA and the nuclear DNA is highly conserved in land plants, but a notable exception is rpl21. This gene has been found in the completely sequenced chloroplast genome of a lower plant but not in that of two higher plants. We describe the purification and characterization of the spinach chloroplast ribosomal protein L21 and the isolation and nucleotide sequence of a cDNA clone that encodes its cytoplasmic precursor. The mature protein, identified by NH2-terminal sequencing, has 201 residues (Mr 22,766) and is thus substantially larger than either its Escherichia coli (103 residues) or the lower plant homologue (116 residues). The extra length is in peptide extensions at both amino and carboxyl termini. The COOH-terminal extension is unusual in that it comprises seven Ala-Glu repeats, a feature not found in any other ribosomal proteins described so far. The cDNA clone also encodes a 55-residue long transit peptide (with a high proportion of the polar residues, threonine and serine), to target the L21 protein into chloroplasts. The identification of rpl21 as a nuclear gene in a higher plant (spinach) and chloroplast gene in a lower plant (liverwort) suggests an organelle-to-nucleus gene relocation during the evolution of the former.     

The Mitochondrial Genome of Soybean Reveals Complex Genome Structures and Gene Evolution at Intercellular and Phylogenetic Levels

Determining mitochondrial genomes is important for elucidating vital activities of seed plants. Mitochondrial genomes are specific to each plant species because of their variable size, complex structures and patterns of gene losses and gains during evolution. This complexity has made research on the soybean mitochondrial genome difficult compared with its nuclear and chloroplast genomes. The present study helps to solve a 30-year mystery regarding the most complex mitochondrial genome structure, showing that pairwise rearrangements among the many large repeats may produce an enriched molecular pool of 760 circles in seed plants. The soybean mitochondrial genome harbors 58 genes of known function in addition to 52 predicted open reading frames of unknown function. The genome contains sequences of multiple identifiable origins, including 6.8 kb and 7.1 kb DNA fragments that have been transferred from the nuclear and chloroplast genomes, respectively, and some horizontal DNA transfers. The soybean mitochondrial genome has lost 16 genes, including nine protein-coding genes and seven tRNA genes; however, it has acquired five chloroplast-derived genes during evolution. Four tRNA genes, common among the three genomes, are derived from the chloroplast. Sizeable DNA transfers to the nucleus, with pericentromeric regions as hotspots, are observed, including DNA transfers of 125.0 kb and 151.6 kb identified unambiguously from the soybean mitochondrial and chloroplast genomes, respectively. The soybean nuclear genome has acquired five genes from its mitochondrial genome. These results provide biological insights into the mitochondrial genome of seed plants, and are especially helpful for deciphering vital activities in soybean.     

The tetrapyrrole synthesis pathway as a model of horizontal gene transfer in euglenoids

The history of euglenoids may have begun as early as ~2 bya. These early phagotrophs ate cyanobacteria, archaea, and eubacteria, and the subsequent appearance of red algae and chromalveolates provided euglenoids with additional food sources. Following the appearance of green algae, euglenoids acquired a chloroplast via a secondary endosymbiotic event with a green algal ancestor. This endosymbiosis also involved a massive transfer of nuclear‐encoded genes from the symbiont nucleus to the host. Expecting these genes to have a green algal origin, this research has shown, through the use of DNA‐sequences and the analysis of phylogenetic relationships, that many housekeeping genes have a red algal/chromalveolate ancestry. This suggested that many other endosymbiotic/horizontal gene transfers, which brought genes from chromalveolates to euglenoids, may have been taking place long before the acquisition of the chloroplast. The investigation of the origin of the enzymes involved in the tetrapyrrole synthesis pathway provided insights into horizontal gene transfer in euglenoids and demonstrated that the euglenoid nuclear genome is a mosaic comprised of genes from the ancestral lineage plus genes transferred endosymbiotically/horizontally from green, red, and chromalveolates lineages.