Presence of a 16S rRNA pseudogene in the bi-molecular plastid genome of the primitive brown alga Pylaiella littoralis. Evolutionary implications

Summary The plastid genome of the brown alga Pylaiella littoralis (L.) Kjellm. is composed of two different circular DNA molecules: the largest carries two rrn operons, and the smallest, only one copy of both 16S and 23S rDNAs. 16S rDNA copies located on both molecules have been cloned and their nucleotide sequences determined: they are 65% homologous to one another. The expression of these genes was assayed by hybridizing in vivo labelled P. littoralis rRNAs to both clones, and specific oligonucleotides to total RNA from P. littoralis. Results indicate that the 16S rDNA copy located on the small molecule is a pseudogene. Comparisons of the functional gene with other 16S rRNA genes shows that chloroplasts from green plants emerged earlier from the cyanobacterial lineage than Euglena gracilis and Pylaiella littoralis plastids.     

REDUCED CHLOROPLAST COVERAGE genes from Arabidopsis thaliana help to establish the size of the chloroplast compartment

Eukaryotic cells require mechanisms to establish the proportion of cellular volume devoted to particular organelles. These mechanisms are poorly understood. From a screen for plastid-to-nucleus signaling mutants in Arabidopsis thaliana, we cloned a mutant allele of a gene that encodes a protein of unknown function that is homologous to two other Arabidopsis genes of unknown function and to FRIENDLY, which was previously shown to promote the normal distribution of mitochondria in Arabidopsis. In contrast to FRIENDLY, these three homologs of FRIENDLY are found only in photosynthetic organisms. Based on these data, we proposed that FRIENDLY expanded into a small gene family to help regulate the energy metabolism of cells that contain both mitochondria and chloroplasts. Indeed, we found that knocking out these genes caused a number of chloroplast phenotypes, including a reduction in the proportion of cellular volume devoted to chloroplasts to 50% of wild type. Thus, we refer to these genes as REDUCED CHLOROPLAST COVERAGE (REC). The size of the chloroplast compartment was reduced most in rec1 mutants. The REC1 protein accumulated in the cytosol and the nucleus. REC1 was excluded from the nucleus when plants were treated with amitrole, which inhibits cell expansion and chloroplast function. We conclude that REC1 is an extraplastidic protein that helps to establish the size of the chloroplast compartment, and that signals derived from cell expansion or chloroplasts may regulate REC1.Chloroplasts drive plant growth, development, and reproduction by converting solar energy into biologically useful forms of energy. Thus, the biogenesis and function of chloroplasts underpin crop yields and, indeed, life on Earth. Chloroplasts develop from proplastids during germination and leaf development (1). After chloroplast biogenesis, chloroplasts divide by binary fission. A number of mutant alleles enhance or reduce the size of individual chloroplasts by attenuating or promoting chloroplast division (2). Regardless of the size of individual chloroplasts, the proportion of cellular volume devoted to all chloroplasts appears indistinguishable from wild type in these mutants (3–5). Thus, the mechanism that establishes the size of the chloroplast compartment appears distinct from the mechanisms of chloroplast division.The cell expansion that drives the expansion of leaves also drives the proliferation of chloroplasts. Indeed, the proliferation of chloroplasts is so tightly correlated with cell expansion that the ratio of the size of the chloroplast compartment to the size of mesophyll cells is constant, regardless of cell size (2, 6, 7). Cell type exerts a major influence over the proportion of cellular volume devoted to the chloroplast. For instance, the size of the chloroplast compartment in mesophyll cells is larger than in epidermal cells. Thus, an extraplastidic mechanism appears to determine the size of the chloroplast compartment (6). However, during the expansion of leaves, chloroplasts are not completely submissive to the cell. Indeed, chloroplast dysfunction inhibits the expansion of leaves (8).Although mechanisms that establish the proportion of cellular volume devoted to particular organelles are of fundamental importance to biology, these mechanisms remain poorly understood (2). In the particular case of chloroplasts, understanding these mechanisms may lead to significant advances for agriculture. For example, introducing C₄ photosynthesis into rice, a plant that performs C₃ photosynthesis, is one strategy for potentially increasing yields from this important crop (9). C₃ and C₄ leaves are distinct at the metabolic, cellular, and anatomical levels (9, 10). One of the conserved features of C₄ leaves is the increase and decrease in the size of the chloroplast compartment in bundle sheath and mesophyll cells, respectively, relative to C₃ leaves (10). The engineering of C₄ photosynthesis in important C₃ crops, such as rice, is thought to depend on the ability to rationally manipulate the size of the chloroplast compartment (11).We performed a screen for plastid-to-nucleus signaling mutants in Arabidopsis thaliana (12). From this screen, we obtained one mutant allele of a gene that encodes a protein of unknown function. This gene is homologous to two Arabidopsis genes that encode proteins of unknown function and to FRIENDLY. FRIENDLY and its orthologs promote the normal distribution of mitochondria in Arabidopsis and in nonphotosynthetic organisms (13). However, these three Arabidopsis homologs of FRIENDLY are found only in photosynthetic organisms. Based on these data, we thought that FRIENDLY may have expanded into a small gene family to help manage the energy metabolism of cells that contain both chloroplasts and mitochondria. We tested this idea by examining the phenotypes of mutants in which one, two, three, or all four of these genes are knocked out. We found that these mutants exhibited a number of chloroplast phenotypes, including a smaller chloroplast compartment relative to wild type. Thus, we named these genes REDUCED CHLOROPLAST COVERAGE (REC). We also found that the protein that contributes most to establishing the size of the chloroplast compartment, REC1, localizes to both the nucleus and the cytosol, and we provide evidence that signals derived from dysfunctional chloroplasts or the inhibition of cell expansion may regulate the nucleocytoplasmic partitioning of REC1.     

Newly identified and diverse plastid-bearing branch on the eukaryotic tree of life

The use of molecular methods is altering our understanding of the microbial biosphere and the complexity of the tree of life. Here, we report a newly discovered uncultured plastid-bearing eukaryotic lineage named the rappemonads. Phylogenies using near-complete plastid ribosomal DNA (rDNA) operons demonstrate that this group represents an evolutionarily distinct lineage branching with haptophyte and cryptophyte algae. Environmental DNA sequencing revealed extensive diversity at North Atlantic, North Pacific, and European freshwater sites, suggesting a broad ecophysiology and wide habitat distribution. Quantitative PCR analyses demonstrate that the rappemonads are often rare but can form transient blooms in the Sargasso Sea, where high 16S rRNA gene copies mL(-1) were detected in late winter. This pattern is consistent with these microbes being a member of the rare biosphere, whose constituents have been proposed to play important roles under ecosystem change. Fluorescence in situ hybridization revealed that cells from this unique lineage were 6.6 ± 1.2 × 5.7 ± 1.0 μm, larger than numerically dominant open-ocean phytoplankton, and appear to contain two to four plastids. The rappemonads are unique, widespread, putatively photosynthetic algae that are absent from present-day ecosystem models and current versions of the tree of life.     

Cryo-EM study of the spinach chloroplast ribosome reveals the structural and functional roles of plastid-specific ribosomal proteins

Protein synthesis in the chloroplast is carried out by chloroplast ribosomes (chloro-ribosome) and regulated in a light-dependent manner. Chloroplast or plastid ribosomal proteins (PRPs) generally are larger than their bacterial counterparts, and chloro-ribosomes contain additional plastid-specific ribosomal proteins (PSRPs); however, it is unclear to what extent these proteins play structural or regulatory roles during translation. We have obtained a three-dimensional cryo-EM map of the spinach 70S chloro-ribosome, revealing the overall structural organization to be similar to bacterial ribosomes. Fitting of the conserved portions of the x-ray crystallographic structure of the bacterial 70S ribosome into our cryo-EM map of the chloro-ribosome reveals the positions of PRP extensions and the locations of the PSRPs. Surprisingly, PSRP1 binds in the decoding region of the small (30S) ribosomal subunit, in a manner that would preclude the binding of messenger and transfer RNAs to the ribosome, suggesting that PSRP1 is a translation factor rather than a ribosomal protein. PSRP2 and PSRP3 appear to structurally compensate for missing segments of the 16S rRNA within the 30S subunit, whereas PSRP4 occupies a position buried within the head of the 30S subunit. One of the two PSRPs in the large (50S) ribosomal subunit lies near the tRNA exit site. Furthermore, we find a mass of density corresponding to chloro-ribosome recycling factor; domain II of this factor appears to interact with the flexible C-terminal domain of PSRP1. Our study provides evolutionary insights into the structural and functional roles that the PSRPs play during protein synthesis in chloroplasts.     

Genome-wide analyses of Geraniaceae plastid DNA reveal unprecedented patterns of increased nucleotide substitutions

Angiosperm plastid genomes are generally conserved in gene content and order with rates of nucleotide substitutions for protein-coding genes lower than for nuclear protein-coding genes. A few groups have experienced genomic change, and extreme changes in gene content and order are found within the flowering plant family Geraniaceae. The complete plastid genome sequence of Pelargonium X hortorum (Geraniaceae) reveals the largest and most rearranged plastid genome identified to date. Highly elevated rates of sequence evolution in Geraniaceae mitochondrial genomes have been reported, but rates in Geraniaceae plastid genomes have not been characterized. Analysis of nucleotide substitution rates for 72 plastid genes for 47 angiosperm taxa, including nine Geraniaceae, show that values of dN are highly accelerated in ribosomal protein and RNA polymerase genes throughout the family. Furthermore, dN/dS is significantly elevated in the same two classes of plastid genes as well as in ATPase genes. A relatively high dN/dS ratio could be interpreted as evidence of two phenomena, namely positive or relaxed selection, neither of which is consistent with our current understanding of plastid genome evolution in photosynthetic plants. These analyses are the first to use protein-coding sequences from complete plastid genomes to characterize rates and patterns of sequence evolution for a broad sampling of photosynthetic angiosperms, and they reveal unprecedented accumulation of nucleotide substitutions in Geraniaceae. To explain these remarkable substitution patterns in the highly rearranged Geraniaceae plastid genomes, we propose a model of aberrant DNA repair coupled with altered gene expression.     

Trafficking of protein into the recently established photosynthetic organelles of Paulinella chromatophora

Endosymbiotic acquisition of bacteria by a protist, with subsequent evolution of the bacteria into mitochondria and plastids, had a transformative impact on eukaryotic biology. Reconstructing events that created a stable association between endosymbiont and host during the process of organellogenesis--including establishment of regulated protein import into nascent organelles--is difficult because they date back more than 1 billion years. The amoeba Paulinella chromatophora contains nascent photosynthetic organelles of more recent evolutionary origin (～60 Mya) termed chromatophores (CRs). After the initial endosymbiotic event, the CR genome was reduced to approximately 30% of its presumed original size and more than 30 expressed genes were transferred from the CR to the amoebal nuclear genome. Three transferred genes--psaE, psaK1, and psaK2--encode subunits of photosystem I. Here we report biochemical evidence that PsaE, PsaK1, and PsaK2 are synthesized in the amoeba cytoplasm and traffic into CRs, where they assemble with CR-encoded subunits into photosystem I complexes. Additionally, our data suggest that proteins routed to CRs pass through the Golgi apparatus. Whereas genome reduction and transfer of genes from bacterial to host genome have been reported to occur in other obligate bacterial endosymbioses, this report outlines the import of proteins encoded by such transferred genes into the compartment derived from the bacterial endosymbiont. Our study showcases P. chromatophora as an exceptional model in which to study early events in organellogenesis, and suggests that protein import into bacterial endosymbionts might be a phenomenon much more widespread than currently assumed.     

Cell-to-cell movement of mitochondria in plants

We report cell-to-cell movement of mitochondria through a graft junction. Mitochondrial movement was discovered in an experiment designed to select for chloroplast transfer from Nicotiana sylvestris into Nicotiana tabacum cells. The alloplasmic N. tabacum line we used carries Nicotiana undulata cytoplasmic genomes, and its flowers are male sterile due to the foreign mitochondrial genome. Thus, rare mitochondrial DNA transfer from N. sylvestris to N. tabacum could be recognized by restoration of fertile flower anatomy. Analyses of the mitochondrial genomes revealed extensive recombination, tentatively linking male sterility to orf293, a mitochondrial gene causing homeotic conversion of anthers into petals. Demonstrating cell-to-cell movement of mitochondria reconstructs the evolutionary process of horizontal mitochondrial DNA transfer and enables modification of the mitochondrial genome by DNA transmitted from a sexually incompatible species. Conversion of anthers into petals is a visual marker that can be useful for mitochondrial transformation.Horizontal gene transfer (HGT), the acquisition of gene(s) across species mating boundaries, results in a phylogeny of the transferred gene(s) that is incongruent with the phylogeny of the organism. In flowering plants, HGT is relatively rare in the nucleus, but frequently involves mitochondrial DNA (mtDNA); for reviews see refs. 1–3. Pioneering papers described HGT of several mitochondrial genes (4–6) and, in an extreme case, incorporation of six genome equivalents in the 3.9-Mb Amborella trichopoda mtDNA (7). These findings imply that mechanisms exist for DNA delivery between unrelated species. Parasitic plants are frequent participants in HGT, either as donors (8, 9) or recipients (5) of foreign DNA; the DNA exchange between the host and parasite is probably facilitated by the physical connection (for review, see ref. 3). HGT between nonparasites, however, necessitates alternative modes of DNA transfer. Transfer via vectoring agents such as viruses, bacteria, fungi, insects, and pollen; transformational uptake of plant DNA released into the soil; and occasional grafting of unrelated species were proposed (6). The first experimental evidence in support of grafting as a potential mechanism of HGT came from demonstrating exchange of plant DNA in tobacco tissue grafts (10). Movement of entire chloroplast genomes was subsequently demonstrated through tobacco graft junctions, interpreted as evidence for cell-to-cell movement of the organelles (11, 12). However, evidence for cell-to-cell movement of mitochondrial DNA is missing in plants despite the fact that the majority of horizontal gene transfer events involve mitochondrial sequences.We report here an experimental system for the successful identification of a rare mitochondrial HGT event. Replacing the cytoplasm of Nicotiana tabacum with the cytoplasm of Nicotiana undulata makes the flowers of N. tabacum male sterile due to conversion of anthers to stigmatoid petals (Fig. 1 D–G). Such N. tabacum plants are called alloplasmic substitution lines for carrying an alien cytoplasm and are cytoplasmic male sterile (CMS) because they inherit male sterility only from the maternal parent (13). We reasoned that movement of Nicotiana sylvestris mitochondria into CMS cells should restore anther morphology and pollen production, a change that is easy to detect in plants even if restricted to a few flowers.Open in a separate windowFig. 1.Restoration of fertile flower anatomy facilitates identification of mitochondrial graft transmission event. (A) N. tabacum Nt-CMS and fertile N. sylvestris Ns-F graft partners and GT19-C seed progeny. (B) Grafting tobacco in culture. The scion is Nt-CMS, which carries the nuclear gentamycin resistance marker; and the rootstock is Ns-F, which carries the plastid spectinomycin resistance (aadA) and aurea bar^au genes. Arrow points to graft junction. (C) Selection of gentamycin and spectinomycin double-resistant clones. (Right) Stem slices from the graft region; (Left) from above and below. Arrow points to double-resistant clone. (D) One isolated anther from a wild-type N. tabacum flower (above) and the anther after homeotic conversion of the N. tabacum alloplasmic substitution line (below). (E) Flower morphology of the graft partners and mixed flower anatomy on the GT19-C graft transmission plant. (Right) Flowers are shown with corolla; (Left) with corolla removed. Note homeotic transformation of anthers into stigmatoid petals in Nt-CMS graft partner and the GT-CMS flowers. GT-F and N. sylvestris Ns-F flowers are fertile. The flowers of Nt-CMS graft partner and GT19-C plant (GT-CMS and GT-F) are pink, a nuclear trait; those of the N. sylvestris graft partner are white. A close-up of (F) GT-CMS, (G) GT-intermediate, and (H) GT-F flowers. (Scale bars in Lower Right corners, 10 mm.)We looked for cell-to-cell movement of mitochondria in stem grafts of two species, N. tabacum and N. sylvestris. We first selected for the nuclear marker from N. tabacum and the chloroplast marker in N. sylvestris and regenerated plants from double-resistant tissue derived from the graft junction. We identified branches with fertile flowers on one of the regenerated plants, indicating presence of fertile mtDNA in the otherwise CMS plant, and analyzed the mtDNA of its fertile and CMS seed progeny. Recombination at alternative sites in the mitochondrial genome facilitated the identification of a candidate mitochondrial gene responsible for homeotic transformation of anthers resulting in CMS.     

Synthesis and degradation of dinoflagellate plastid-encoded psbA proteins are light-regulated, not circadian-regulated

In many dinoflagellate species, the plastid genome has been proposed to exist as a limited number of single-gene minicircles, and many genes normally found in the plastid genome are nuclear-encoded. Unlike the nuclear-encoded plastid-directed gene products whose expression is often regulated by the circadian clock, little is known about expression of minicircle genes. Furthermore, even the plastid location of the minicircles has recently been challenged. We have examined the incorporation in vivo of [(35)S]methionine into the proteins of purified plastids, and we find that several plastid proteins are labeled in the presence of cycloheximide but not chloramphenicol. One of these proteins, labeled in two different dinoflagellate species, was identified as psbA by Western blot analysis. Furthermore, this psbA has the expected physiological characteristics, because both synthesis and degradation are induced by light. We find no evidence for circadian control over either synthesis or degradation of psbA, unlike the several nuclear-encoded plastid-directed proteins studied. Finally, we find that levels of psbA protein or RNA do not change over a 24-h light-dark cycle, suggesting that this protein may not be involved in mediating the circadian rhythm in oxygen evolution rates. This demonstration is the first, to our knowledge, that minicircle genes encoding plastid proteins are translated in dinoflagellate plastids, and it suggests that a proteomic approach to characterizing the dinoflagellate plastid genome is feasible.