Cryo-EM structures of the plant plastid-encoded RNA polymerase.

Chloroplasts are green plastids in the cytoplasm of eukaryotic algae and plants responsible for photosynthesis. The plastid-encoded RNA polymerase (PEP) plays an essential role during chloroplast biogenesis from proplastids and functions as the predominant RNA polymerase in mature chloroplasts. The PEP-centered transcription apparatus comprises a bacterial-origin PEP core and more than a dozen eukaryotic-origin PEP-associated proteins (PAPs) encoded in the nucleus. Here, we determined the cryo-EM structures of Nicotiana tabacum (tobacco) PEP-PAP apoenzyme and PEP-PAP transcription elongation complexes at near-atomic resolutions. Our data show the PEP core adopts a typical fold as bacterial RNAP. Fifteen PAPs bind at the periphery of the PEP core, facilitate assembling the PEP-PAP supercomplex, protect the complex from oxidation damage, and likely couple gene transcription with RNA processing. Our results report the high-resolution architecture of the chloroplast transcription apparatus and provide the structural basis for the mechanistic and functional study of transcription regulation in chloroplasts.

Structure of the plant plastid-encoded RNA polymerase.

Chloroplast genes encoding photosynthesis-associated proteins are predominantly transcribed by the plastid-encoded RNA polymerase (PEP). PEP is a multi-subunit complex composed of plastid-encoded subunits similar to bacterial RNA polymerases (RNAPs) stably bound to a set of nuclear-encoded PEP-associated proteins (PAPs). PAPs are essential to PEP activity and chloroplast biogenesis, but their roles are poorly defined. Here, we present cryoelectron microscopy (cryo-EM) structures of native 21-subunit PEP and a PEP transcription elongation complex from white mustard (Sinapis alba). We identify that PAPs encase the core polymerase, forming extensive interactions that likely promote complex assembly and stability. During elongation, PAPs interact with DNA downstream of the transcription bubble and with the nascent mRNA. The models reveal details of the superoxide dismutase, lysine methyltransferase, thioredoxin, and amino acid ligase enzymes that are subunits of PEP. Collectively, these data provide a foundation for the mechanistic understanding of chloroplast transcription and its role in plant growth and adaptation.

An RNA polymerase that became a Swiss army knife.

RNA polymerases (RNAPs) control the first step of gene expression in all forms of life by transferring genetic information from DNA to RNA, a process known as transcription. In this issue of Cell, Webster et al. and Wu et al. report three-dimensional structures of RNAP complexes from chloroplasts.

Amyloplast sedimentation repolarizes LAZYs to achieve gravity sensing in plants.

Gravity controls directional growth of plants, and the classical starch-statolith hypothesis proposed more than a century ago postulates that amyloplast sedimentation in specialized cells initiates gravity sensing, but the molecular mechanism remains uncharacterized. The LAZY proteins are known as key regulators of gravitropism, and lazy mutants show striking gravitropic defects. Here, we report that gravistimulation by reorientation triggers mitogen-activated protein kinase (MAPK) signaling-mediated phosphorylation of Arabidopsis LAZY proteins basally polarized in root columella cells. Phosphorylation of LAZY increases its interaction with several translocons at the outer envelope membrane of chloroplasts (TOC) proteins on the surface of amyloplasts, facilitating enrichment of LAZY proteins on amyloplasts. Amyloplast sedimentation subsequently guides LAZY to relocate to the new lower side of the plasma membrane in columella cells, where LAZY induces asymmetrical auxin distribution and root differential growth. Together, this study provides a molecular interpretation for the starch-statolith hypothesis: the organelle-movement-triggered molecular polarity formation.

Structure of the multi-subunit chloroplast RNA polymerase.

Chloroplasts contain a dedicated genome that encodes subunits of the photosynthesis machinery. Transcription of photosynthesis genes is predominantly carried out by a plastid-encoded RNA polymerase (PEP), a nearly 1 MDa complex composed of core subunits with homology to eubacterial RNA polymerases (RNAPs) and at least 12 additional chloroplast-specific PEP-associated proteins (PAPs). However, the architecture of this complex and the functions of the PAPs remain unknown. Here, we report the cryo-EM structure of a 19-subunit PEP complex from Sinapis alba (white mustard). The structure reveals that the PEP core resembles prokaryotic and nuclear RNAPs but contains chloroplast-specific features that mediate interactions with the PAPs. The PAPs are unrelated to known transcription factors and arrange around the core in a unique fashion. Their structures suggest potential functions during transcription in the chemical environment of chloroplasts. These results reveal structural insights into chloroplast transcription and provide a framework for understanding photosynthesis gene expression.

Complementary environmental analysis and functional characterization of lower glycolysis-gluconeogenesis in the diatom plastid.

Organic carbon fixed in chloroplasts through the Calvin-Benson-Bassham Cycle can be diverted toward different metabolic fates, including cytoplasmic and mitochondrial respiration, gluconeogenesis, and synthesis of diverse plastid metabolites via the pyruvate hub. In plants, pyruvate is principally produced via cytoplasmic glycolysis, although a plastid-targeted lower glycolytic pathway is known to exist in non-photosynthetic tissue. Here, we characterized a lower plastid glycolysis-gluconeogenesis pathway enabling the direct interconversion of glyceraldehyde-3-phosphate and phospho-enol-pyruvate in diatoms, ecologically important marine algae distantly related to plants. We show that two reversible enzymes required to complete diatom plastid glycolysis-gluconeogenesis, Enolase and bis-phosphoglycerate mutase (PGAM), originated through duplications of mitochondria-targeted respiratory isoforms. Through CRISPR-Cas9 mutagenesis, integrative 'omic analyses, and measured kinetics of expressed enzymes in the diatom Phaeodactylum tricornutum, we present evidence that this pathway diverts plastid glyceraldehyde-3-phosphate into the pyruvate hub, and may also function in the gluconeogenic direction. Considering experimental data, we show that this pathway has different roles dependent in particular on day length and environmental temperature, and show that the cpEnolase and cpPGAM genes are expressed at elevated levels in high-latitude oceans where diatoms are abundant. Our data provide evolutionary, meta-genomic, and functional insights into a poorly understood yet evolutionarily recurrent plastid metabolic pathway.

Polyploid plants take cytonuclear perturbations in stride.

Hybridization in plants is often accompanied by nuclear genome doubling (allopolyploidy), which has been hypothesized to perturb interactions between nuclear and organellar (mitochondrial and plastid) genomes by creating imbalances in the relative copy number of these genomes and producing genetic incompatibilities between maternally derived organellar genomes and the half of the allopolyploid nuclear genome from the paternal progenitor. Several evolutionary responses have been predicted to ameliorate these effects, including selection for changes in protein sequences that restore cytonuclear interactions; biased gene retention/expression/conversion favoring maternal nuclear gene copies; and fine-tuning of relative cytonuclear genome copy numbers and expression levels. Numerous recent studies, however, have found that evolutionary responses are inconsistent and rarely scale to genome-wide generalities. The apparent robustness of plant cytonuclear interactions to allopolyploidy may reflect features that are general to allopolyploids such as the lack of F2 hybrid breakdown under disomic inheritance, and others that are more plant-specific, including slow sequence divergence in organellar genomes and preexisting regulatory responses to changes in cell size and endopolyploidy during development. Thus, cytonuclear interactions may only rarely act as the main barrier to establishment of allopolyploid lineages, perhaps helping to explain why allopolyploidy is so pervasive in plant evolution.

Plastid retrograde signaling: A developmental perspective.

Chloroplast activities influence nuclear gene expression, a phenomenon referred to as retrograde signaling. Biogenic retrograde signals have been revealed by changes in nuclear gene expression when chloroplast development is disrupted. Research on biogenic signaling has focused on repression of Photosynthesis-Associated Nuclear Genes (PhANGs), but this is just one component of a syndrome involving altered expression of thousands of genes involved in diverse processes, many of which are upregulated. We discuss evidence for a framework that accounts for most of this syndrome. Disruption of chloroplast biogenesis prevents the production of signals required to progress through discrete steps in the program of photosynthetic differentiation, causing retention of juvenile states. As a result, expression of PhANGs and other genes that act late during photosynthetic differentiation is not initiated, while expression of genes that act early is retained. The extent of juvenility, and thus the transcriptome, reflects the disrupted process: lack of plastid translation blocks development very early, whereas disruption of photosynthesis without compromising plastid translation blocks development at a later stage. We discuss implications of these and other recent observations for the nature of the plastid-derived signals that regulate photosynthetic differentiation and the role of GUN1, an enigmatic protein involved in biogenic signaling.

Identification of a highly efficient chloroplast-targeting peptide for plastid engineering.

Plastids are pivotal target organelles for comprehensively enhancing photosynthetic and metabolic traits in plants via plastid engineering. Plastidial proteins predominantly originate in the nucleus and must traverse membrane-bound multiprotein translocons to access these organelles. This import process is meticulously regulated by chloroplast-targeting peptides (cTPs). Whereas many cTPs have been employed to guide recombinantly expressed functional proteins to chloroplasts, there is a critical need for more efficient cTPs. Here, we performed a comprehensive exploration and comparative assessment of an advanced suite of cTPs exhibiting superior targeting capabilities. We employed a multifaceted approach encompassing computational prediction, in planta expression, fluorescence tracking, and in vitro chloroplast import studies to identify and analyze 88 cTPs associated with Arabidopsis thaliana mutants with phenotypes linked to chloroplast function. These polypeptides exhibited distinct abilities to transport green fluorescent protein (GFP) to various compartments within leaf cells, particularly chloroplasts. A highly efficient cTP derived from Arabidopsis plastid ribosomal protein L35 (At2g24090) displayed remarkable effectiveness in chloroplast localization. This cTP facilitated the activities of chloroplast-targeted RNA-processing proteins and metabolic enzymes within plastids. This cTP could serve as an ideal transit peptide for precisely targeting biomolecules to plastids, leading to advancements in plastid engineering.

A molecular atlas of plastid and mitochondrial proteins reveals organellar remodeling during plant evolutionary transitions from algae to angiosperms.

Algae and plants carry 2 organelles of endosymbiotic origin that have been co-evolving in their host cells for more than a billion years. The biology of plastids and mitochondria can differ significantly across major lineages and organelle changes likely accompanied the adaptation to new ecological niches such as the terrestrial habitat. Based on organelle proteome data and the genomes of 168 phototrophic (Archaeplastida) versus a broad range of 518 non-phototrophic eukaryotes, we screened for changes in plastid and mitochondrial biology across 1 billion years of evolution. Taking into account 331,571 protein families (or orthogroups), we identify 31,625 protein families that are unique to primary plastid-bearing eukaryotes. The 1,906 and 825 protein families are predicted to operate in plastids and mitochondria, respectively. Tracing the evolutionary history of these protein families through evolutionary time uncovers the significant remodeling the organelles experienced from algae to land plants. The analyses of gained orthogroups identifies molecular changes of organelle biology that connect to the diversification of major lineages and facilitated major transitions from chlorophytes en route to the global greening and origin of angiosperms.

Retrograde signaling by the plastidial metabolite MEcPP regulates expression of nuclear stress-response genes.

Plastid-derived signals are known to coordinate expression of nuclear genes encoding plastid-localized proteins in a process termed retrograde signaling. To date, the identity of retrograde-signaling molecules has remained elusive. Here, we show that methylerythritol cyclodiphosphate (MEcPP), a precursor of isoprenoids produced by the plastidial methylerythritol phosphate (MEP) pathway, elicits the expression of selected stress-responsive nuclear-encoded plastidial proteins. Genetic and pharmacological manipulations of the individual MEP pathway metabolite levels demonstrate the high specificity of MEcPP as an inducer of these targeted stress-responsive genes. We further demonstrate that abiotic stresses elevate MEcPP levels, eliciting the expression of the aforementioned genes. We propose that the MEP pathway, in addition to producing isoprenoids, functions as a stress sensor and a coordinator of expression of targeted stress-responsive nuclear genes via modulation of the levels of MEcPP, a specific and critical retrograde-signaling metabolite.

Pyrenoid proteomics reveals independent evolution of the CO2-concentrating organelle in chlorarachniophytes.

Pyrenoids are microcompartments that are universally found in the photosynthetic plastids of various eukaryotic algae. They contain ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and play a pivotal role in facilitating CO2 assimilation via CO2-concentrating mechanisms (CCMs). Recent investigations involving model algae have revealed that pyrenoid-associated proteins participate in pyrenoid biogenesis and CCMs. However, these organisms represent only a small part of algal lineages, which limits our comprehensive understanding of the diversity and evolution of pyrenoid-based CCMs. Here we report a pyrenoid proteome of the chlorarachniophyte alga Amorphochlora amoebiformis, which possesses complex plastids acquired through secondary endosymbiosis with green algae. Proteomic analysis using mass spectrometry resulted in the identification of 154 potential pyrenoid components. Subsequent localization experiments demonstrated the specific targeting of eight proteins to pyrenoids. These included a putative Rubisco-binding linker, carbonic anhydrase, membrane transporter, and uncharacterized GTPase proteins. Notably, most of these proteins were unique to this algal lineage. We suggest a plausible scenario in which pyrenoids in chlorarachniophytes have evolved independently, as their components are not inherited from green algal pyrenoids.

Maternal plastid inheritance: two abating factors identified.

Organelle DNAs (orgDNAs) in mitochondria and plastids are generally inherited from the maternal parent; however, it is unclear how their inheritance mode is controlled, particularly in the plastids of seed plants. Chung et al. identify two factors that affect maternal inheritance in tobacco plastids: cold temperature and DNA amount in pollen.

Plastid dsRNA transgenes trigger phased small RNA-based gene silencing of nuclear-encoded genes.

Plastid transformation technology has been widely used to express traits of potential commercial importance, though the technology has been limited to traits that function while sequestered in the organelle. Prior research indicates that plastid contents can escape from the organelle, suggesting a possible mechanism for engineering plastid transgenes to function in other cellular locations. To test this hypothesis, we created tobacco (Nicotiana tabacum cv. Petit Havana) plastid transformants that express a fragment of the nuclear-encoded Phytoene desaturase (PDS) gene capable of catalyzing post-transcriptional gene silencing if RNA escapes into the cytoplasm. We found multiple lines of direct evidence that plastid-encoded PDS transgenes affect nuclear PDS gene silencing: knockdown of the nuclear-encoded PDS mRNA and/or its apparent translational inhibition, biogenesis of 21-nucleotide (nt) phased small interfering RNAs (phasiRNAs), and pigment-deficient plants. Furthermore, plastid-expressed dsRNA with no cognate nuclear-encoded pairing partner also produced abundant 21-nt phasiRNAs in the cytoplasm, demonstrating that a nuclear-encoded template is not required for siRNA biogenesis. Our results indicate that RNA escape from plastids to the cytoplasm occurs generally, with functional consequences that include entry into the gene silencing pathway. Furthermore, we uncover a method to produce plastid-encoded traits with functions outside of the organelle and open additional fields of study in plastid development, compartmentalization, and small RNA biogenesis.

Initiation of B-type starch granules in wheat endosperm requires the plastidial α-glucan phosphorylase PHS1.

The plastidial α-glucan phosphorylase (PHS1) can elongate and degrade maltooligosaccharides (MOSs), but its exact physiological role in plants is poorly understood. Here, we discover a specialized role of PHS1 in establishing the unique bimodal characteristic of starch granules in wheat (Triticum spp.) endosperm. Wheat endosperm contains large A-type granules that initiate at early grain development and small B-type granules that initiate in later grain development. We demonstrate that PHS1 interacts with B-GRANULE CONTENT1 (BGC1), a carbohydrate-binding protein essential for normal B-type granule initiation. Mutants of tetraploid durum wheat (Triticum turgidum) deficient in all homoeologs of PHS1 had normal A-type granules but fewer and larger B-type granules. Grain size and starch content were not affected by the mutations. Further, by assessing granule numbers during grain development in the phs1 mutant and using a double mutant defective in both PHS1 and BGC1, we demonstrate that PHS1 is exclusively involved in B-type granule initiation. The total starch content and number of starch granules per chloroplast in leaves were not affected by loss of PHS1, suggesting that its role in granule initiation in wheat is limited to the endosperm. We therefore propose that the initiation of A- and B-type granules occurs via distinct biochemical mechanisms, where PHS1 plays an exclusive role in B-type granule initiation.

A phase-separated CO2-fixing pyrenoid proteome determined by TurboID in Chlamydomonas reinhardtii.

Phase separation underpins many biologically important cellular events such as RNA metabolism, signaling, and CO2 fixation. However, determining the composition of a phase-separated organelle is often challenging due to its sensitivity to environmental conditions, which limits the application of traditional proteomic techniques like organellar purification or affinity purification mass spectrometry to understand their composition. In Chlamydomonas reinhardtii, Rubisco is condensed into a crucial phase-separated organelle called the pyrenoid that improves photosynthetic performance by supplying Rubisco with elevated concentrations of CO2. Here, we developed a TurboID-based proximity labeling technique in which proximal proteins in Chlamydomonas chloroplasts are labeled by biotin radicals generated from the TurboID-tagged protein. By fusing 2 core pyrenoid components with the TurboID tag, we generated a high-confidence pyrenoid proxiome that contains most known pyrenoid proteins, in addition to new pyrenoid candidates. Fluorescence protein tagging of 7 previously uncharacterized TurboID-identified proteins showed that 6 localized to a range of subpyrenoid regions. The resulting proxiome also suggests new secondary functions for the pyrenoid in RNA-associated processes and redox-sensitive iron-sulfur cluster metabolism. This developed pipeline can be used to investigate a broad range of biological processes in Chlamydomonas, especially at a temporally resolved suborganellar resolution.

The pyrenoid: the eukaryotic CO2-concentrating organelle.

The pyrenoid is a phase-separated organelle that enhances photosynthetic carbon assimilation in most eukaryotic algae and the land plant hornwort lineage. Pyrenoids mediate approximately one-third of global CO2 fixation, and engineering a pyrenoid into C3 crops is predicted to boost CO2 uptake and increase yields. Pyrenoids enhance the activity of the CO2-fixing enzyme Rubisco by supplying it with concentrated CO2. All pyrenoids have a dense matrix of Rubisco associated with photosynthetic thylakoid membranes that are thought to supply concentrated CO2. Many pyrenoids are also surrounded by polysaccharide structures that may slow CO2 leakage. Phylogenetic analysis and pyrenoid morphological diversity support a convergent evolutionary origin for pyrenoids. Most of the molecular understanding of pyrenoids comes from the model green alga Chlamydomonas (Chlamydomonas reinhardtii). The Chlamydomonas pyrenoid exhibits multiple liquid-like behaviors, including internal mixing, division by fission, and dissolution and condensation in response to environmental cues and during the cell cycle. Pyrenoid assembly and function are induced by CO2 availability and light, and although transcriptional regulators have been identified, posttranslational regulation remains to be characterized. Here, we summarize the current knowledge of pyrenoid function, structure, components, and dynamic regulation in Chlamydomonas and extrapolate to pyrenoids in other species.

The evolution of strictly monofunctional naphthoquinol C-methyltransferases is vital in cyanobacteria and plastids.

Prenylated quinones are membrane-associated metabolites that serve as vital electron carriers for respiration and photosynthesis. The UbiE (EC 2.1.1.201)/MenG (EC 2.1.1.163) C-methyltransferases catalyze pivotal ring methylations in the biosynthetic pathways of many of these quinones. In a puzzling evolutionary pattern, prokaryotic and eukaryotic UbiE/MenG homologs segregate into 2 clades. Clade 1 members occur universally in prokaryotes and eukaryotes, excluding cyanobacteria, and include mitochondrial COQ5 enzymes required for ubiquinone biosynthesis; Clade 2 members are specific to cyanobacteria and plastids. Functional complementation of an Escherichia coli ubiE/menG mutant indicated that Clade 1 members display activity with both demethylbenzoquinols and demethylnaphthoquinols, independently of the quinone profile of their original taxa, while Clade 2 members have evolved strict substrate specificity for demethylnaphthoquinols. Expression of the gene-encoding bifunctional Arabidopsis (Arabidopsis thaliana) COQ5 in the cyanobacterium Synechocystis or its retargeting to Arabidopsis plastids resulted in synthesis of a methylated variant of plastoquinone-9 that does not occur in nature. Accumulation of methylplastoquinone-9 was acutely cytotoxic, leading to the emergence of suppressor mutations in Synechocystis and seedling lethality in Arabidopsis. These data demonstrate that in cyanobacteria and plastids, co-occurrence of phylloquinone and plastoquinone-9 has driven the evolution of monofunctional demethylnaphthoquinol methyltransferases and explains why plants cannot capture the intrinsic bifunctionality of UbiE/MenG to simultaneously synthesize their respiratory and photosynthetic quinones.

Arabidopsis EXECUTER1 interacts with WRKY transcription factors to mediate plastid-to-nucleus singlet oxygen signaling.

Chloroplasts produce singlet oxygen (1O2), which causes changes in nuclear gene expression through plastid-to-nucleus retrograde signaling to increase plant fitness. However, the identity of this 1O2-triggered pathway remains unclear. Here, we identify mutations in GENOMES UNCOUPLED4 (GUN4) and GUN5 as suppressors of phytochrome-interacting factor1 (pif1) pif3 in regulating the photo-oxidative response in Arabidopsis thaliana. GUN4 and GUN5 specifically interact with EXECUTER1 (EX1) and EX2 in plastids, and this interaction is alleviated by treatment with Rose Bengal (RB) or white light. Impaired expression of GUN4, GUN5, EX1, or EX2 leads to insensitivity to excess light and overexpression of EX1 triggers photo-oxidative responses. Strikingly, upon light irradiation or RB treatment, EX1 transiently accumulates in the nucleus and the nuclear fraction of EX1 shows a similar molecular weight as the plastid-located protein. Point mutagenesis analysis indicated that nuclear localization of EX1 is required for its function. EX1 acts as a transcriptional co-activator and interacts with the transcription factors WRKY18 and WRKY40 to promote the expression of 1O2-responsive genes. This study suggests that EX1 may act in plastid-to-nucleus signaling and establishes a 1O2-triggered retrograde signaling pathway that allows plants adapt to changing light environments during chloroplast development.

Components of iron-Sulfur cluster assembly machineries are robust phylogenetic markers to trace the origin of mitochondria and plastids.

Establishing the origin of mitochondria and plastids is key to understand 2 founding events in the origin and early evolution of eukaryotes. Recent advances in the exploration of microbial diversity and in phylogenomics approaches have indicated a deep origin of mitochondria and plastids during the diversification of Alphaproteobacteria and Cyanobacteria, respectively. Here, we strongly support these placements by analyzing the machineries for assembly of iron-sulfur ([Fe-S]) clusters, an essential function in eukaryotic cells that is carried out in mitochondria by the ISC machinery and in plastids by the SUF machinery. We assessed the taxonomic distribution of ISC and SUF in representatives of major eukaryotic supergroups and analyzed the phylogenetic relationships with their prokaryotic homologues. Concatenation datasets of core ISC proteins show an early branching of mitochondria within Alphaproteobacteria, right after the emergence of Magnetococcales. Similar analyses with the SUF machinery place primary plastids as sister to Gloeomargarita within Cyanobacteria. Our results add to the growing evidence of an early emergence of primary organelles and show that the analysis of essential machineries of endosymbiotic origin provide a robust signal to resolve ancient and fundamental steps in eukaryotic evolution.