Chloroplast genomes in Populus (Salicaceae): comparisons from an intensively sampled genus reveal dynamic patterns of evolution

The chloroplast is one of two organelles containing a separate genome that codes for essential and distinct cellular functions such as photosynthesis. Given the importance of chloroplasts in plant metabolism, the genomic architecture and gene content have been strongly conserved through long periods of time and as such are useful molecular tools for evolutionary inferences. At present, complete chloroplast genomes from over 4000 species have been deposited into publicly accessible databases. Despite the large number of complete chloroplast genomes, comprehensive analyses regarding genome architecture and gene content have not been conducted for many lineages with complete species sampling. In this study, we employed the genus Populus to assess how more comprehensively sampled chloroplast genome analyses can be used in understanding chloroplast evolution in a broadly studied lineage of angiosperms. We conducted comparative analyses across Populus in order to elucidate variation in key genome features such as genome size, gene number, gene content, repeat type and number, SSR (Simple Sequence Repeat) abundance, and boundary positioning between the four main units of the genome. We found that some genome annotations were variable across the genus owing in part from errors in assembly or data checking and from this provided corrected annotations. We also employed complete chloroplast genomes for phylogenetic analyses including the dating of divergence times throughout the genus. Lastly, we utilized re-sequencing data to describe the variations of pan-chloroplast genomes at the population level for P. euphratica. The analyses used in this paper provide a blueprint for the types of analyses that can be conducted with publicly available chloroplast genomes as well as methods for building upon existing datasets to improve evolutionary inference.

Outer membrane-deprived cyanobacteria liberate periplasmic and thylakoid luminal components that support the growth of heterotrophs

ABSTRACTChloroplasts originate from endosymbiosis of a cyanobacterium within a heterotrophic host cell. Establishing endosymbiosis requires the translocation across its envelope of photosynthetic products generated inside the once free-living cyanobacterium to be exploited by host metabolism. However, the nature of this translocation event is unknown. We previously found that most cyanobacterial outer membrane components were eliminated during the primitive stage of chloroplast evolution, suggesting the importance of evolutionary changes of the outer membrane. Here, we removed the outer membrane from Synechocystis sp. PCC 6803 by disrupting the physical interaction with peptidoglycan, and characterized the effects on cell function. Outer membrane-deprived cells liberated diverse substances into the environment without significantly compromising photoautotrophic growth. The amount of liberated proteins increased to ~0.35 g/L within five days of culture. Proteomic analysis showed that most liberated proteins were periplasmic and thylakoid luminal components. Connectivity between the thylakoid lumen-extracellular space was confirmed by findings that an exogenous hydrophilic oxidant was reduced by photosynthetic electron transport chain on the thylakoid membrane. Metabolomic analysis detected the release of nucleotide-related metabolites at concentrations around 1 μM. The liberated materials supported the proliferation of heterotrophic bacteria. These findings show that breaching the outer membrane, without any manipulations to the cytoplasmic membrane, converts a cyanobacterium to a chloroplast-like organism that conducts photosynthesis and releases its biogenic materials. This conversion not only represents a potential explanation why the outer membrane markedly changed during the earliest stage of chloroplast evolution, but also provides the opportunity to harness cyanobacterial photosynthesis for biomanufacturing processes.SIGNIFICANCE STATEMENTAlthough it is well accepted that chloroplasts stem from endosymbiosis of a cyanobacterium within a heterotrophic host cell, the issue of how photosynthetic products generated inside a formerly free-living cyanobacterium are translocated across its envelope and exploited by host metabolism has been little addressed. Here we show that breaching the cyanobacterial outer membrane barrier converts a cyanobacterium to a chloroplast-like organism that conducts photosynthesis and releases its diverse biogenic materials into its external environment, which sustains the growth of heterotrophic organisms. This conversion represents a possible example of metabolic exploitation of cyanobacterial photosynthesis. Further, this “quasi-chloroplast” provides a potential opportunity for industrial application such as producing feedstock for biomanufacturing processes that harnesses heterotrophic bacteria.

Functional Analysis of PSRP1, the Chloroplast Homolog of a Cyanobacterial Ribosome Hibernation Factor

Bacterial ribosome hibernation factors sequester ribosomes in an inactive state during the stationary phase and in response to stress. The cyanobacterial ribosome hibernation factor LrtA has been suggested to inactivate ribosomes in the dark and to be important for post-stress survival. In this study, we addressed the hypothesis that Plastid Specific Ribosomal Protein 1 (PSRP1), the chloroplast-localized LrtA homolog in plants, contributes to the global repression of chloroplast translation that occurs when plants are shifted from light to dark. We found that the abundance of PSRP1 and its association with ribosomes were similar in the light and the dark. Maize mutants lacking PSRP1 were phenotypically normal under standard laboratory growth conditions. Furthermore, the absence of PSRP1 did not alter the distribution of chloroplast ribosomes among monosomes and polysomes in the light or in the dark, and did not affect the light-regulated synthesis of the chloroplast psbA gene product. These results suggest that PSRP1 does not play a significant role in the regulation of chloroplast translation by light. As such, the physiological driving force for the retention of PSRP1 during chloroplast evolution remains unclear.

Unveiling the hidden genetic diversity and chloroplast type of marine benthic ciliate Mesodinium species

Ciliate Mesodinium species are commonly distributed in diverse aquatic systems worldwide. Among Mesodinium species, M. rubrum is closely associated with microbial food webs and red tide formation and is known to acquire chloroplasts from its cryptophyte prey for use in photosynthesis. For these reasons, Mesodinium has long received much attention in terms of ecophysiology and chloroplast evolution. Mesodinium cells are easily identifiable from other organisms owing to their unique morphology comprising two hemispheres, but a clear distinction among species is difficult under a microscope. Recent taxonomic studies of Mesodinium have been conducted largely in parallel with molecular sequence analysis, and the results have shown that the best-known planktonic M. rubrum in fact comprises eight genetic clades of a M. rubrum/M. major complex. However, unlike the planktonic Mesodinium species, little is known of the genetic diversity of benthic Mesodinium species, and to our knowledge, the present study is the first to explore this. A total of ten genetic clades, including two clades composed of M. chamaeleon and M. coatsi, were found in marine sandy sediments, eight of which were clades newly discovered through this study. We report the updated phylogenetic relationship within the genus Mesodinium comprising heterotrophic/mixotrophic as well as planktonic/benthic species. Furthermore, we unveiled the wide variety of chloroplasts of benthic Mesodinium, which were related to the green cryptophyte Chroomonas/Hemiselmis and the red cryptophyte Rhodomonas/Storeatula/Teleaulax groups.