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

Abstract

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.

Figure 1.

Losses of Chloroplast infA during Angiosperm Evolution Based on Analysis of Sequenced infA Loci. Shown are all angiosperms whose chloroplast infA loci have been sequenced, except that only one representative of the 17 sequenced Solanaceae species is shown. Black bars indicate lineages with chloroplast infA pseudogenes or complete loss. The branching order and taxonomic classifications are based on Soltis et al. (1999) and Olmstead et al. (2000). Boxed names indicate species from which a nuclear infA gene was identified.

Figure 2.

Evidence for Loss of Chloroplast infA from DNA Gel Blot Hybridization. The three probes named at top were hybridized sequentially to a filter containing BamHI digests of total DNA from 280 angiosperms, 96 of which are shown here. Note that some infA genes have a BamHI site near their middle. SSU, small subunit.

Figure 3.

Losses of Chloroplast infA during Angiosperm Evolution Based on DNA Gel Blot Hybridization Results. The infA probes used were both from Antirrhinum. Plants with significantly diminished or no hybridization are shaded (see Figure 2 and text). Plant names beside the tree alternate in two columns. Loss lineages are shown in boldface on the tree and are marked with bullets. Bull's-eye bullets indicate the eight loss lineages for which loss was confirmed by sequencing of chloroplast infA from at least one member of the lineage. Within rosids, the sequenced member(s) was included on the blots; for the other four sequence-validated loss lineages, a different member of the loss lineage was sequenced than was on the blots. In particular, Tricyrtis (Figure 2) is the sister taxon to Smilax relative to all other taxa on the blots, Caltha is likewise sister to the Clematis-Ranunculus clade, Pentas is sister to Galium, and Campanula belongs within the Lobelia-Trachelium clade.

Figure 4.

Multiple Alignment of IF1 Protein Sequences from the Four Nuclear infA Genes and Two Representative Chloroplast Genes. The alignment was made using ClustalW 1.8 (Thompson et al., 1994), with the most common residue at each position highlighted. Predicted chloroplast transit peptides are shown by lowercase letters, and their predicted cleavage sites (Emanuelsson et al., 1999) are marked by large X symbols. The alignment of the transit peptides is not meant to indicate that they share a common ancestor (see text). cp, chloroplast; nuc, nuclear.

Figure 5.

Localization of Fusion Proteins Encoded by Nuclear InfA-gfp Constructs to Chloroplasts. The soybean InfA-gfp and Arabidopsis InfA-gfp were introduced into epidermal cells of tobacco and Arabidopsis using microprojectile bombardment. The localization of GFP in individual transformed cells was detected by confocal laser scanning microscopy 3 days after bombardment. (A) to (C) pGmInfA-gfp in a tobacco guard cell. The images show two guard cells; the upper cell shows expression of pGmInfA-gfp. GFP fluorescence (A). Chlorophyll fluorescence (B). Merged images (C). Bar in (C) = 5 μm. (D) to (F) Arabidopsis epidermal pavement cells with one cell expressing pAtInfA-gfp. The chloroplasts in epidermal pavement cells are smaller than those in guard cells. GFP fluorescence (D). Chlorophyll fluorescence (E). Merged images (F). Bar in (F) = 10 μm.

Figure 6.

Phylogenetic Analysis of Nuclear and Chloroplast infA Sequences. The diagram summarizes the maximum likelihood trees found when each of the four nuclear infA sequences was added individually to a constrained topology (the species phylogeny from Figure 1) for the 21 chloroplast sequences. The arrows indicate the most likely placement of the tomato (Solanum), soybean (Glycine), ice plant (Mesembryanthemum), and Arabidopsis nuclear sequences, and all branch lengths are drawn to scale horizontally. Mesembryanthemum cp denotes the ice plant chloroplast pseudogene. The branch lengths for the chloroplast sequences were identical in the four trees, allowing them to be merged. cp, chloroplast; nuc, nuclear.