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Current Opinion in Biotechnology Oct 2019Plastids are interesting targets for metabolic engineering using the tools of synthetic biology. Plastids carry their own genome, which can be manipulated genetically in... (Review)
Review
Plastids are interesting targets for metabolic engineering using the tools of synthetic biology. Plastids carry their own genome, which can be manipulated genetically in many algae and plants. Incorporating foreign genes into the plastid genome offers valuable benefits, such as high-level foreign protein expression and the absence of gene silencing. Here, we review progress in bioengineering of chloroplasts to produce valuable metabolites and proteins. Various strategies for enhancing yields of desired products, including design of operons, fusion proteins for improved translational efficiency, protein scaffolding, metabolic channeling and storage, are described. Efforts to control plastid differentiation also offer promising ways of turning plastids into controllable bio-factories, and the construction of synthetic plastids optimized for specific functions would be a major advance.
Topics: Chloroplasts; Metabolic Engineering; Plants, Genetically Modified; Plastids; Synthetic Biology
PubMed: 30798145
DOI: 10.1016/j.copbio.2019.01.009 -
Proceedings of the National Academy of... Jun 2022The evolution of eukaryotic life was predicated on the development of organelles such as mitochondria and plastids. During this complex process of organellogenesis, the...
The evolution of eukaryotic life was predicated on the development of organelles such as mitochondria and plastids. During this complex process of organellogenesis, the host cell and the engulfed prokaryote became genetically codependent, with the integration of genes from the endosymbiont into the host nuclear genome and subsequent gene loss from the endosymbiont. This process required that horizontally transferred genes become active and properly regulated despite inherent differences in genetic features between donor (endosymbiont) and recipient (host). Although this genetic reorganization is considered critical for early stages of organellogenesis, we have little knowledge about the mechanisms governing this process. The photosynthetic amoeba Paulinella micropora offers a unique opportunity to study early evolutionary events associated with organellogenesis and primary endosymbiosis. This amoeba harbors a “chromatophore,” a nascent photosynthetic organelle derived from a relatively recent cyanobacterial association (∼120 million years ago) that is independent of the evolution of primary plastids in plants (initiated ∼1.5 billion years ago). Analysis of the genome and transcriptome of Paulinella revealed that retrotransposition of endosymbiont-derived nuclear genes was critical for their domestication in the host. These retrocopied genes involved in photoprotection in cyanobacteria became expanded gene families and were “rewired,” acquiring light-responsive regulatory elements that function in the host. The establishment of host control of endosymbiont-derived genes likely enabled the cell to withstand photo-oxidative stress generated by oxygenic photosynthesis in the nascent organelle. These results provide insights into the genetic mechanisms and evolutionary pressures that facilitated the metabolic integration of the host–endosymbiont association and sustained the evolution of a photosynthetic organelle.
Topics: Amoeba; Biological Evolution; Eukaryota; Plastids; Rhizaria; Symbiosis
PubMed: 35639693
DOI: 10.1073/pnas.2121241119 -
Current Protocols Feb 2023Plastids are found in all plant cell types. However, most extraction methods to study these organelles are performed at the organ level (e.g., leaf, root, fruit) and do...
Plastids are found in all plant cell types. However, most extraction methods to study these organelles are performed at the organ level (e.g., leaf, root, fruit) and do not allow for tissue-specific resolution, which hinders our understanding of their physiology. Therefore, IPTACT (Isolation of Plastids TAgged in specific Cell Types) was developed to isolate plastids in a tissue-specific manner in Arabidopsis thaliana (Arabidopsis). Plastids are biotinylated using one-shot transgenic lines, and tissue specificity is achieved with a suitable promoter as long as such a promoter exists. Cell-specific biotinylated plastids are then isolated with 2.8-µm streptavidin beads. Plastids extracted by IPTACT are suitable for RNA or protein isolation and subsequent tissue-specific OMICs analyses. This method provides the user with a powerful tool to investigate plastidial functions at cell-type resolution. Furthermore, it can easily be combined with studies using diverse genetic backgrounds and/or different developmental or stress conditions. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Promoter cloning and plant selection Basic Protocol 2: Isolation of biotinylated plastids Basic Protocol 3: Quality control of isolated plastids.
Topics: Arabidopsis; Plastids; Arabidopsis Proteins; Promoter Regions, Genetic; Fruit
PubMed: 36799650
DOI: 10.1002/cpz1.673 -
Methods in Enzymology 2022Carotenoid biosynthesis and sequestration in higher plants occurs in the plastid organelle. Among diverse germplasm collections displaying natural variation for...
Carotenoid biosynthesis and sequestration in higher plants occurs in the plastid organelle. Among diverse germplasm collections displaying natural variation for carotenoids and outputs from metabolic engineering experiments it has become clear that plastid type and numbers can have important implications on the quantitative composition of carotenoids accumulating. Therefore, it is important to characterize these organelles to fully evaluate the potential of the germplasm to enhance carotenoids and create nutrient dense fruits and vegetables. In this article the procedures used to isolate sub-plastidial structures from carotenoid-rich Solanaceae fruits (tomato and Capsicum) are described.
Topics: Carotenoids; Fruit; Gene Expression Regulation, Plant; Solanum lycopersicum; Plastids
PubMed: 35878982
DOI: 10.1016/bs.mie.2022.03.001 -
Planta Oct 2023In this study, we assembled the complete plastome and mitogenome of Caragana spinosa and explored the multiple configurations of the organelle genomes. Caragana spinosa...
In this study, we assembled the complete plastome and mitogenome of Caragana spinosa and explored the multiple configurations of the organelle genomes. Caragana spinosa belongs to the Papilionoidea subfamily and has significant pharmaceutical value. To explore the possible interaction between the organelle genomes, we assembled and analyzed the plastome and mitogenome of C. spinosa using the Illumina and Nanopore DNA sequencing data. The plastome of C. spinosa was 129,995 bp belonging to the inverted repeat lacking clade (IRLC), which contained 77 protein-coding genes, 29 tRNA genes, and four rRNA genes. The mitogenome was 378,373 bp long and encoded 54 unique genes, including 33 protein-coding, three ribosomal RNA (rRNA), and 18 transfer RNA (tRNA) genes. In addition to the single circular conformation, alternative conformations mediated by one and four repetitive sequences in the plastome and mitogenome were identified and validated, respectively. The inverted repeat (PDR12, the 12th dispersed repeat sequence in C. spinosa plastome) of plastome mediating recombinant was conserved in the genus Caragana. Furthermore, we identified 14 homologous fragments by comparing the sequences of mitogenome and plastome, including eight complete tRNA genes. A phylogenetic analysis of protein-coding genes extracted from the plastid and mitochondrial genomes revealed congruent topologies. Analyses of sequence divergence found one intergenic region, trnN-GUU-ycf1, exhibiting a high degree of variation, which can be used to develop novel molecular markers to distinguish the nine Caragana species accurately. This plastome and mitogenome of C. spinosa could provide critical information for the molecular breeding of C. spinosa and be used as a reference genome for other species of Caragana. In this study, we assembled the complete plastome and mitogenome of Caragana spinosa and explored the multiple configurations of the organelle genomes.
Topics: Genome, Mitochondrial; Caragana; Phylogeny; Plastids; RNA, Transfer; Genome, Plastid
PubMed: 37831319
DOI: 10.1007/s00425-023-04245-6 -
Plant Cell Reports Apr 2022Global survey of plastid gene expression during fruit ripening in kiwifruit provides cis-elements for the future engineering of the plastid genome of kiwifruit. A...
Global survey of plastid gene expression during fruit ripening in kiwifruit provides cis-elements for the future engineering of the plastid genome of kiwifruit. A limitation in the application of plastid biotechnology for molecular farming is the low-level expression of transgenes in non-green plastids compared with photosynthetically active chloroplasts. Unlike other fruits, not all chloroplasts are transformed into chromoplasts during ripening of red-fleshed kiwifruit (Actinidia chinensis cv. Hongyang) fruits, which may make kiwifruit an ideal horticultural plant for recombinant protein production by plastid engineering. To identify cis-elements potentially triggering high-level transgene expression in edible tissues of the 'Hongyang' kiwifruit, here we report a comprehensive analysis of kiwifruit plastid gene transcription in green leaves and fruits at three different developmental stages. While transcripts of a few photosynthesis-related genes and most genetic system genes were substantially upregulated in green fruits compared with leaves, nearly all plastid genes were significantly downregulated at the RNA level during fruit development. Expression of a few genes remained unchanged, including psbA, the gene encoding the D1 polypeptide of photosystem II. However, PsbA protein accumulation decreased continuously during chloroplast-to-chromoplast differentiation. Analysis of post-transcriptional steps in mRNA maturation, including intron splicing and RNA editing, revealed that splicing and editing may contribute to regulation of plastid gene expression. Altogether, 40 RNA editing sites were verified, and 5 of them were newly discovered. Taken together, this study has generated a valuable resource for the analysis of plastid gene expression and provides cis-elements for future efforts to engineer the plastid genome of kiwifruit.
Topics: Actinidia; Fruit; Gene Expression Regulation, Plant; Plant Proteins; Plastids; Transcription, Genetic
PubMed: 35226116
DOI: 10.1007/s00299-022-02840-7 -
BioMed Research International 2021Species of (Liliaceae) are of great horticultural importance and are distributed across Europe, North Africa, and Asia. The Tien Shan Mountain is one of the primary...
Species of (Liliaceae) are of great horticultural importance and are distributed across Europe, North Africa, and Asia. The Tien Shan Mountain is one of the primary diversity centres of , but the molecular studies of species from this location are lacking. In our study, we assembled four plastid genomes from the Tien Shan Mountains, , , , and , combined with the plastid genome of to compare against other Liliaceae plastid genomes. We focussed on the species diversity and evolution of their plastid genomes. The five plastid genomes proved highly similar in overall size (151,691-152,088 bp), structure, gene order, and content. With comparative analysis, we chose 7 mononucleotide SSRs from the species that could be used in further population studies. Phylogenetic analyses based on 24 plastid genomes robustly supported the monophyly of and the sister relationship between and , . , , and were clustered together, and was clustered with , with our results clearly demonstrating the relationships between these five species. Our results provide a more comprehensive understanding of the phylogenomics and comparative genomics of .
Topics: Biological Evolution; Codon; DNA, Plant; Evolution, Molecular; Gene Order; Genome, Plastid; Genomics; Liliaceae; Microsatellite Repeats; Nucleotides; Phylogeny; Plastids; Polymorphism, Single Nucleotide; Tulipa
PubMed: 34239930
DOI: 10.1155/2021/6648429 -
Molecular Biology and Evolution Apr 2022Ochrophyta is an algal group belonging to the Stramenopiles and comprises diverse lineages of algae which contribute significantly to the oceanic ecosystems as primary...
Ochrophyta is an algal group belonging to the Stramenopiles and comprises diverse lineages of algae which contribute significantly to the oceanic ecosystems as primary producers. However, early evolution of the plastid organelle in Ochrophyta is not fully understood. In this study, we provide a well-supported tree of the Stramenopiles inferred by the large-scale phylogenomic analysis that unveils the eukaryvorous (nonphotosynthetic) protist Actinophrys sol (Actinophryidae) is closely related to Ochrophyta. We used genomic and transcriptomic data generated from A. sol to detect molecular traits of its plastid and we found no evidence of plastid genome and plastid-mediated biosynthesis, consistent with previous ultrastructural studies that did not identify any plastids in Actinophryidae. Moreover, our phylogenetic analyses of particular biosynthetic pathways provide no evidence of a current and past plastid in A. sol. However, we found more than a dozen organellar aminoacyl-tRNA synthases (aaRSs) that are of algal origin. Close relationships between aaRS from A. sol and their ochrophyte homologs document gene transfer of algal genes that happened before the divergence of Actinophryidae and Ochrophyta lineages. We further showed experimentally that organellar aaRSs of A. sol are targeted exclusively to mitochondria, although organellar aaRSs in Ochrophyta are dually targeted to mitochondria and plastids. Together, our findings suggested that the last common ancestor of Actinophryidae and Ochrophyta had not yet completed the establishment of host-plastid partnership as seen in the current Ochrophyta species, but acquired at least certain nuclear-encoded genes for the plastid functions.
Topics: Ecosystem; Evolution, Molecular; Genome, Plastid; Phylogeny; Plants; Plastids; Stramenopiles
PubMed: 35348760
DOI: 10.1093/molbev/msac065 -
Current Biology : CB Sep 2023Plastid symbioses between heterotrophic hosts and algae are widespread and abundant in surface oceans. They are critically important both for extant ecological systems...
Plastid symbioses between heterotrophic hosts and algae are widespread and abundant in surface oceans. They are critically important both for extant ecological systems and for understanding the evolution of plastids. Kleptoplastidy, where the plastids of prey are temporarily retained and continuously re-acquired, provides opportunities to study the transitional states of plastid establishment. Here, we investigated the poorly studied marine centrohelid Meringosphaera and its previously unidentified symbionts using culture-independent methods from environmental samples. Investigations of the 18S rDNA from single-cell assembled genomes (SAGs) revealed uncharacterized genetic diversity within Meringosphaera that likely represents multiple species. We found that Meringosphaera harbors plastids of Dictyochophyceae origin (stramenopiles), for which we recovered six full plastid genomes and found evidence of two distinct subgroups that are congruent with host identity. Environmental monitoring by qPCR and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) revealed seasonal dynamics of both host and plastid. In particular, we did not detect the plastids for 6 months of the year, which, combined with the lack of plastids in some SAGs, suggests that the plastids are temporary and the relationship is kleptoplastidic. Importantly, we found evidence of genetic integration of the kleptoplasts as we identified host-encoded plastid-associated genes, with evolutionary origins likely from the plastid source as well as from other alga sources. This is only the second case where host-encoded kleptoplast-targeted genes have been predicted in an ancestrally plastid-lacking group. Our results provide evidence for gene transfers and protein re-targeting as relatively early events in the evolution of plastid symbioses.
Topics: Symbiosis; In Situ Hybridization, Fluorescence; Genome; Plastids; Phylogeny
PubMed: 37536342
DOI: 10.1016/j.cub.2023.07.017 -
Proceedings of the National Academy of... Mar 2022SignificanceAlthough plastid division is critical for plant development, how components of the plastid division machinery (PDM) are imported into plastids remains...
SignificanceAlthough plastid division is critical for plant development, how components of the plastid division machinery (PDM) are imported into plastids remains unexplored. A forward genetic screen to identify suppressors of a () mutant deficient in plastid division led us to find dominant gain-of-function (GF) mutations in , which significantly increases the import of PDM components and completely rescues phenotypes. The defective plastid division phenotypes in and mutants and CRL-TIC236 association in a functional complex indicate that the CRL-TIC236 module is vital for plastid division. Hence, we report the first GF translocon mutants and unveil CRL as a novel functional partner of TIC236 for PDM import.
Topics: Arabidopsis; Arabidopsis Proteins; Cell Division; Chloroplast Proteins; Gain of Function Mutation; Membrane Transport Proteins; Plastids; Protein Transport
PubMed: 35275795
DOI: 10.1073/pnas.2123353119