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.

Structure, function, and assembly of photosystem I in thylakoid membranes of vascular plants.

The photosynthetic apparatus is formed by thylakoid membrane-embedded multiprotein complexes that carry out linear electron transport in oxygenic photosynthesis. The machinery is largely conserved from cyanobacteria to land plants, and structure and function of the protein complexes involved are relatively well studied. By contrast, how the machinery is assembled in thylakoid membranes remains poorly understood. The complexes participating in photosynthetic electron transfer are composed of many proteins, pigments and redox-active cofactors, whose temporally and spatially highly coordinated incorporation is essential to build functional mature complexes. Several proteins, jointly referred to as assembly factors, engage in the biogenesis of these complexes to bring the components together in a step-wise manner, in the right order and time. In this review, we focus on the biogenesis of the terminal protein supercomplex of the photosynthetic electron transport chain, photosystem I (PSI), in vascular plants. We summarize our current knowledge of the assembly process and the factors involved, and describe the challenges associated with resolving the assembly pathway in molecular detail.

CO-EXPRESSED WITH PSI ASSEMBLY1 (CEPA1) is a photosystem I assembly factor in Arabidopsis.

Photosystem I (PSI) forms a large macromolecular complex of ∼580 kDa that resides in the thylakoid membrane and mediates photosynthetic electron transfer. PSI is composed of eighteen protein subunits and nearly two hundred co-factors. The assembly of the complex in thylakoid membranes requires high spatial and temporal coordination, and is critically dependent on a sophisticated assembly machinery. Here, we report and characterize CO-EXPRESSED WITH PSI ASSEMBLY1 (CEPA1), a PSI assembly factor in Arabidopsis (Arabidopsis thaliana). The CEPA1 gene was identified bioinformatically as being co-expressed with known PSI assembly factors. Disruption of the CEPA1 gene leads to a pale phenotype and retarded plant development but does not entirely abolish photoautotrophy. Biophysical and biochemical analyses revealed that the phenotype is caused by a specific defect in PSI accumulation. We further show that CEPA1 acts at the post-translational level and co-localizes with PSI in non-appressed thylakoid membranes. In native gels, CEPA1 co-migrates with thylakoid protein complexes, including putative PSI assembly intermediates. Finally, protein-protein interaction assays suggest cooperation of CEPA1 with the PSI assembly factor PHOTOSYSTEM I ASSEMBLY3 PSA3. Together, our data support an important but non-essential role of CEPA1 in PSI assembly.

Hypothetical chloroplast reading frame 51 encodes a photosystem I assembly factor in cyanobacteria.

Hypothetical chloroplast open reading frames (ycfs) are putative genes in the plastid genomes of photosynthetic eukaryotes. Many ycfs are also conserved in the genomes of cyanobacteria, the presumptive ancestors of present-day chloroplasts. The functions of many ycfs are still unknown. Here, we generated knock-out mutants for ycf51 (sll1702) in the cyanobacterium Synechocystis sp. PCC 6803. The mutants showed reduced photoautotrophic growth due to impaired electron transport between photosystem II (PSII) and PSI. This phenotype results from greatly reduced PSI content in the ycf51 mutant. The ycf51 disruption had little effect on the transcription of genes encoding photosynthetic complex components and the stabilization of the PSI complex. In vitro and in vivo analyses demonstrated that Ycf51 cooperates with PSI assembly factor Ycf3 to mediate PSI assembly. Furthermore, Ycf51 interacts with the PSI subunit PsaC. Together with its specific localization in the thylakoid membrane and the stromal exposure of its hydrophilic region, our data suggest that Ycf51 is involved in PSI complex assembly. Ycf51 is conserved in all sequenced cyanobacteria, including the earliest branching cyanobacteria of the Gloeobacter genus, and is also present in the plastid genomes of glaucophytes. However, Ycf51 has been lost from other photosynthetic eukaryotic lineages. Thus, Ycf51 is a PSI assembly factor that has been functionally replaced during the evolution of oxygenic photosynthetic eukaryotes.

The red alga Porphyridium as a host for molecular farming: Efficient production of immunologically active hepatitis C virus glycoprotein.

Microalgae are promising production platforms for the cost-effective production of recombinant proteins. We have recently established that the red alga Porphyridium purpureum provides superior transgene expression properties, due to the episomal maintenance of transformation vectors as multicopy plasmids in the nucleus. Here, we have explored the potential of Porphyridium to synthesize complex pharmaceutical proteins to high levels. Testing expression constructs for a candidate subunit vaccine against the hepatitis C virus (HCV), we show that the soluble HCV E2 glycoprotein can be produced in transgenic algal cultures to high levels. The antigen undergoes faithful posttranslational modification by N-glycosylation and is recognized by conformationally selective antibodies, suggesting that it adopts a proper antigenic conformation in the endoplasmic reticulum of red algal cells. We also report the experimental determination of the structure of the N-glycan moiety that is attached to glycosylated proteins in Porphyridium. Finally, we demonstrate the immunogenicity of the HCV antigen produced in red algae when administered by injection as pure protein or by feeding of algal biomass.

DNA base editing in nuclear and organellar genomes.

Genome editing continues to revolutionize biological research. Due to its simplicity and flexibility, CRISPR/Cas-based editing has become the preferred technology in most systems. Cas nucleases tolerate fusion to large protein domains, thus allowing combination of their DNA recognition properties with new enzymatic activities. Fusion to nucleoside deaminase or reverse transcriptase domains has produced base editors and prime editors that, instead of generating double-strand breaks in the target sequence, induce site-specific alterations of single (or a few adjacent) nucleotides. The availability of protein-only genome editing reagents based on transcription activator-like effectors has enabled the extension of base editing to the genomes of chloroplasts and mitochondria. In this review, we summarize currently available base editing methods for nuclear and organellar genomes. We highlight recent advances with improving precision, specificity, and efficiency and discuss current limitations and future challenges. We also provide a brief overview of applications in agricultural biotechnology and gene therapy.

Witnessing Genome Evolution: Experimental Reconstruction of Endosymbiotic and Horizontal Gene Transfer.

Present day mitochondria and plastids (chloroplasts) evolved from formerly free-living bacteria that were acquired through endosymbiosis more than a billion years ago. Conversion of the bacterial endosymbionts into cell organelles involved the massive translocation of genetic material from the organellar genomes to the nucleus. The development of transformation technologies for organellar genomes has made it possible to reconstruct this endosymbiotic gene transfer in laboratory experiments and study the mechanisms involved. Recently, the horizontal transfer of genetic information between organisms has also become amenable to experimental investigation. It led to the discovery of horizontal genome transfer as an asexual process generating new species and new combinations of nuclear and organellar genomes. This review describes experimental approaches towards studying endosymbiotic and horizontal gene transfer processes, discusses the new knowledge gained from these approaches about both the evolutionary significance of gene transfer and the underlying molecular mechanisms, and highlights exciting possibilities to exploit gene and genome transfer in biotechnology and synthetic biology.

Chloroplast phosphate transporter CrPHT4-7 regulates phosphate homeostasis and photosynthesis in Chlamydomonas.

In eukaryotic cells, phosphorus is assimilated and utilized primarily as phosphate (Pi). Pi homeostasis is mediated by transporters that have not yet been adequately characterized in green algae. This study reports on PHOSPHATE TRANSPORTER 4-7 (CrPHT4-7) from Chlamydomonas reinhardtii, a member of the PHT4 transporter family, which exhibits remarkable similarity to AtPHT4;4 from Arabidopsis (Arabidopsis thaliana), a chloroplastic ascorbate transporter. Using fluorescent protein tagging, we show that CrPHT4-7 resides in the chloroplast envelope membrane. Crpht4-7 mutants, generated by the CRISPR/Cas12a-mediated single-strand templated repair, show retarded growth, especially in high light, reduced ATP level, strong ascorbate accumulation, and diminished non-photochemical quenching in high light. On the other hand, total cellular phosphorous content was unaffected, and the phenotype of the Crpht4-7 mutants could not be alleviated by ample Pi supply. CrPHT4-7-overexpressing lines exhibit enhanced biomass accumulation under high light conditions in comparison with the wild-type strain. Expressing CrPHT4-7 in a yeast (Saccharomyces cerevisiae) strain lacking Pi transporters substantially recovered its slow growth phenotype, demonstrating that CrPHT4-7 transports Pi. Even though CrPHT4-7 shows a high degree of similarity to AtPHT4;4, it does not display any substantial ascorbate transport activity in yeast or intact algal cells. Thus, the results demonstrate that CrPHT4-7 functions as a chloroplastic Pi transporter essential for maintaining Pi homeostasis and photosynthesis in C. reinhardtii.

Chloroplast translational regulation uncovers nonessential photosynthesis genes as key players in plant cold acclimation.

Plants evolved efficient multifaceted acclimation strategies to cope with low temperatures. Chloroplasts respond to temperature stimuli and participate in temperature sensing and acclimation. However, very little is known about the involvement of chloroplast genes and their expression in plant chilling tolerance. Here we systematically investigated cold acclimation in tobacco seedlings over 2 days of exposure to low temperatures by examining responses in chloroplast genome copy number, transcript accumulation and translation, photosynthesis, cell physiology, and metabolism. Our time-resolved genome-wide investigation of chloroplast gene expression revealed substantial cold-induced translational regulation at both the initiation and elongation levels, in the virtual absence of changes at the transcript level. These cold-triggered dynamics in chloroplast translation are widely distinct from previously described high light-induced effects. Analysis of the gene set responding significantly to the cold stimulus suggested nonessential plastid-encoded subunits of photosynthetic protein complexes as novel players in plant cold acclimation. Functional characterization of one of these cold-responsive chloroplast genes by reverse genetics demonstrated that the encoded protein, the small cytochrome b6f complex subunit PetL, crucially contributes to photosynthetic cold acclimation. Together, our results uncover an important, previously underappreciated role of chloroplast translational regulation in plant cold acclimation.

An RNA thermometer in the chloroplast genome of Chlamydomonas facilitates temperature-controlled gene expression.

Riboregulators such as riboswitches and RNA thermometers provide simple, protein-independent tools to control gene expression at the post-transcriptional level. In bacteria, RNA thermometers regulate protein synthesis in response to temperature shifts. Thermometers outside of the bacterial world are rare, and in organellar genomes, no RNA thermometers have been identified to date. Here we report the discovery of an RNA thermometer in a chloroplast gene of the unicellular green alga Chlamydomonas reinhardtii. The thermometer, residing in the 5' untranslated region of the psaA messenger RNA forms a hairpin-type secondary structure that masks the Shine-Dalgarno sequence at 25°C. At 40°C, melting of the secondary structure increases accessibility of the Shine-Dalgarno sequence to initiating ribosomes, thus enhancing protein synthesis. By targeted nucleotide substitutions and transfer of the thermometer into Escherichia coli, we show that the secondary structure is necessary and sufficient to confer the thermometer properties. We also demonstrate that the thermometer provides a valuable tool for inducible transgene expression from the Chlamydomonas plastid genome, in that a simple temperature shift of the algal culture can greatly increase recombinant protein yields.

Efficient control of western flower thrips by plastid-mediated RNA interference.

Plastid-mediated RNA interference (PM-RNAi) has emerged as a promising strategy for pest control. Expression from the plastid genome of stable double-stranded RNAs (dsRNAs) targeted against essential insect genes can effectively control some herbivorous beetles, but little is known about the efficacy of the transplastomic approach in other groups of pest insects, especially nonchewing insects that do not consume large amounts of leaf material. Here we have investigated the susceptibility of the western flower thrip (WFT, Frankliniella occidentalis), a notorious pest in greenhouses and open fields, to PM-RNAi. We show that WFTs ingest chloroplasts and take up plastid-expressed dsRNAs. We generated a series of transplastomic tobacco plants expressing dsRNAs and hairpin RNAs (hpRNAs) targeted against four essential WFT genes. Unexpectedly, we discovered plastid genome instability in transplastomic plants expressing hpRNAs, suggesting that dsRNA cassettes are preferable over hpRNA cassettes when designing PM-RNAi strategies. Feeding studies revealed that, unlike nuclear transgenic plants, transplastomic plants induced a potent RNAi response in WFTs, causing efficient suppression of the targeted genes and high insect mortality. Our study extends the application range of PM-RNAi technology to an important group of nonchewing insects, reveals design principles for the construction of dsRNA-expressing transplastomic plants, and provides an efficient approach to control one of the toughest insect pests in agriculture and horticulture.

Optimized transgene expression in the red alga Porphyridium purpureum and efficient recombinant protein secretion into the culture medium.

Microalgae represent a promising but yet underexplored production platform for biotechnology. The vast majority of studies on recombinant protein expression in algae have been conducted in a single species, the green alga Chlamydomonas reinhardtii. However, due to epigenetic silencing, transgene expression in Chlamydomonas is often inefficient. Here we have investigated parameters that govern efficient transgene expression in the red microalga Porphyridium purpureum. Porphyridium is unique in that the introduced transformation vectors are episomally maintained as autonomously replicating plasmids in the nucleus. We show that full codon optimization to the preferred codon usage in the Porphyridium genome confers superior transgene expression, not only at the level of protein accumulation, but also at the level of mRNA accumulation, indicating that high translation rates increase mRNA stability. Our optimized expression constructs resulted in YFP accumulation to unprecedented levels of up to 5% of the total soluble protein. We also designed expression cassettes that target foreign proteins to the secretory pathway and lead to efficient protein secretion into the culture medium, thus simplifying recombinant protein harvest and purification. Our study paves the way to the exploration of red microalgae as expression hosts in molecular farming for recombinant proteins and metabolites.

A heat-inducible expression system for external control of gene expression in plastids.

Inducible expression systems can overcome the trade-off between high-level transgene expression and its pleiotropic effects on plant growth. In addition, they can facilitate the expression of biochemical pathways that produce toxic metabolites. Although a few inducible expression systems for the control of transgene expression in plastids have been developed, they all depend on chemical inducers and/or nuclear transgenes. Here we report a temperature-inducible expression system for plastids that is based on the bacteriophage λ leftward and rightward promoters (pL/pR) and the temperature-sensitive repressor cI857. We show that the expression of green fluorescent protein (GFP) in plastids can be efficiently repressed by cI857 under normal growth conditions, and becomes induced over time upon exposure to elevated temperatures in a light-dependent process. We further demonstrate that by introducing into plastids an expression system based on the bacteriophage T7 RNA polymerase, the temperature-dependent accumulation of GFP increased further and was ~24 times higher than expression driven by the pL/pR promoter alone, reaching ~0.48% of the total soluble protein. In conclusion, our heat-inducible expression system provides a new tool for the external control of plastid (trans) gene expression that is cost-effective and does not depend on chemical inducers.

Characterization of mutants deficient in N-terminal phosphorylation of the chloroplast ATP synthase subunit ß.

Understanding the regulation of photosynthetic light harvesting and electron transfer is of great importance to efforts to improve the ability of the electron transport chain to supply downstream metabolism. A central regulator of the electron transport chain is ATP synthase, the molecular motor that harnesses the chemiosmotic potential generated from proton-coupled electron transport to synthesize ATP. ATP synthase is regulated both thermodynamically and post-translationally, with proposed phosphorylation sites on multiple subunits. In this study we focused on two N-terminal serines on the catalytic subunit ß in tobacco (Nicotiana tabacum), previously proposed to be important for dark inactivation of the complex to avoid ATP hydrolysis at night. Here we show that there is no clear role for phosphorylation in the dark inactivation of ATP synthase. Instead, mutation of one of the two phosphorylated serine residues to aspartate to mimic constitutive phosphorylation strongly decreased ATP synthase abundance. We propose that the loss of N-terminal phosphorylation of ATPß may be involved in proper ATP synthase accumulation during complex assembly.

Eukaryote-specific assembly factor DEAP2 mediates an early step of photosystem II assembly in Arabidopsis.

The initial step of oxygenic photosynthesis is the thermodynamically challenging extraction of electrons from water and the release of molecular oxygen. This light-driven process, which is the basis for most life on Earth, is catalyzed by photosystem II (PSII) within the thylakoid membrane of photosynthetic organisms. The biogenesis of PSII requires a controlled step-wise assembly process of which the early steps are considered to be highly conserved between plants and their cyanobacterial progenitors. This assembly process involves auxiliary proteins, which are likewise conserved. In the present work, we used Arabidopsis (Arabidopsis thaliana) as a model to show that in plants, a eukaryote-exclusive assembly factor facilitates the early assembly step, during which the intrinsic antenna protein CP47 becomes associated with the PSII reaction center (RC) to form the RC47 intermediate. This factor, which we named DECREASED ELECTRON TRANSPORT AT PSII (DEAP2), works in concert with the conserved PHOTOSYNTHESIS AFFECTED MUTANT 68 (PAM68) assembly factor. The deap2 and pam68 mutants showed similar defects in PSII accumulation and assembly of the RC47 intermediate. The combined lack of both proteins resulted in a loss of functional PSII and the inability of plants to grow photoautotrophically on the soil. While overexpression of DEAP2 partially rescued the pam68 PSII accumulation phenotype, this effect was not reciprocal. DEAP2 accumulated at 20-fold higher levels than PAM68, together suggesting that both proteins have distinct functions. In summary, our results uncover eukaryotic adjustments to the PSII assembly process, which involve the addition of DEAP2 for the rapid progression from RC to RC47.

GUN control in retrograde signaling: How GENOMES UNCOUPLED proteins adjust nuclear gene expression to plastid biogenesis.

Communication between cellular compartments is vital for development and environmental adaptation. Signals emanating from organelles, so-called retrograde signals, coordinate nuclear gene expression with the developmental stage and/or the functional status of the organelle. Plastids (best known in their green photosynthesizing differentiated form, the chloroplasts) are the primary energy-producing compartment of plant cells, and the site for the biosynthesis of many metabolites, including fatty acids, amino acids, nucleotides, isoprenoids, tetrapyrroles, vitamins, and phytohormone precursors. Signals derived from plastids regulate the accumulation of a large set of nucleus-encoded proteins, many of which localize to plastids. A set of mutants defective in retrograde signaling (genomes uncoupled, or gun) was isolated over 25 years ago. While most GUN genes act in tetrapyrrole biosynthesis, resolving the molecular function of GUN1, the proposed integrator of multiple retrograde signals, has turned out to be particularly challenging. Based on its amino acid sequence, GUN1 was initially predicted to be a plastid-localized nucleic acid-binding protein. Only recently, mechanistic information on the function of GUN1 has been obtained, pointing to a role in plastid protein homeostasis. This review article summarizes our current understanding of GUN-related retrograde signaling and provides a critical appraisal of the various proposed roles for GUNs and their respective pathways.

A photosynthesis operon in the chloroplast genome drives speciation in evening primroses.

Genetic incompatibility between the cytoplasm and the nucleus is thought to be a major factor in species formation, but mechanistic understanding of this process is poor. In evening primroses (Oenothera spp.), a model plant for organelle genetics and population biology, hybrid offspring regularly display chloroplast-nuclear incompatibility. This usually manifests in bleached plants, more rarely in hybrid sterility or embryonic lethality. Hence, most of these incompatibilities affect photosynthetic capability, a trait that is under selection in changing environments. Here we show that light-dependent misregulation of the plastid psbB operon, which encodes core subunits of photosystem II and the cytochrome b6f complex, can lead to hybrid incompatibility, and this ultimately drives speciation. This misregulation causes an impaired light acclimation response in incompatible plants. Moreover, as a result of their different chloroplast genotypes, the parental lines differ in photosynthesis performance upon exposure to different light conditions. Significantly, the incompatible chloroplast genome is naturally found in xeric habitats with high light intensities, whereas the compatible one is limited to mesic habitats. Consequently, our data raise the possibility that the hybridization barrier evolved as a result of adaptation to specific climatic conditions.

Correction of frameshift mutations in the atpB gene by translational recoding in chloroplasts of Oenothera and tobacco.

Translational recoding, also known as ribosomal frameshifting, is a process that causes ribosome slippage along the messenger RNA, thereby changing the amino acid sequence of the synthesized protein. Whether the chloroplast employs recoding is unknown. I-iota, a plastome mutant of Oenothera (evening primrose), carries a single adenine insertion in an oligoA stretch [11A] of the atpB coding region (encoding the ß-subunit of the ATP synthase). The mutation is expected to cause synthesis of a truncated, nonfunctional protein. We report that a full-length AtpB protein is detectable in I-iota leaves, suggesting operation of a recoding mechanism. To characterize the phenomenon, we generated transplastomic tobacco lines in which the atpB reading frame was altered by insertions or deletions in the oligoA motif. We observed that insertion of two adenines was more efficiently corrected than insertion of a single adenine, or deletion of one or two adenines. We further show that homopolymeric composition of the oligoA stretch is essential for recoding, as an additional replacement of AAA lysine codon by AAG resulted in an albino phenotype. Our work provides evidence for the operation of translational recoding in chloroplasts. Recoding enables correction of frameshift mutations and can restore photoautotrophic growth in the presence of a mutation that otherwise would be lethal.

Curvature thylakoid 1 proteins modulate prolamellar body morphology and promote organized thylakoid biogenesis in Arabidopsis thaliana.

The term "de-etiolation" refers to the light-dependent differentiation of etioplasts to chloroplasts in angiosperms. The underlying process involves reorganization of prolamellar bodies (PLBs) and prothylakoids into thylakoids, with concurrent changes in protein, lipid, and pigment composition, which together lead to the assembly of active photosynthetic complexes. Despite the highly conserved structure of PLBs among land plants, the processes that mediate PLB maintenance and their disassembly during de-etiolation are poorly understood. Among chloroplast thylakoid membrane-localized proteins, to date, only Curvature thylakoid 1 (CURT1) proteins were shown to exhibit intrinsic membrane-bending capacity. Here, we show that CURT1 proteins, which play a critical role in grana margin architecture and thylakoid plasticity, also participate in de-etiolation and modulate PLB geometry and density. Lack of CURT1 proteins severely perturbs PLB organization and vesicle fusion, leading to reduced accumulation of the light-dependent enzyme protochlorophyllide oxidoreductase (LPOR) and a delay in the onset of photosynthesis. In contrast, overexpression of CURT1A induces excessive bending of PLB membranes, which upon illumination show retarded disassembly and concomitant overaccumulation of LPOR, though without affecting greening or the establishment of photosynthesis. We conclude that CURT1 proteins contribute to the maintenance of the paracrystalline PLB morphology and are necessary for efficient and organized thylakoid membrane maturation during de-etiolation.

Emergence of Novel RNA-Editing Sites by Changes in the Binding Affinity of a Conserved PPR Protein.

RNA editing converts cytidines to uridines in plant organellar transcripts. Editing typically restores codons for conserved amino acids. During evolution, specific C-to-U editing sites can be lost from some plant lineages by genomic C-to-T mutations. By contrast, the emergence of novel editing sites is less well documented. Editing sites are recognized by pentatricopeptide repeat (PPR) proteins with high specificity. RNA recognition by PPR proteins is partially predictable, but prediction is often inadequate for PPRs involved in RNA editing. Here we have characterized evolution and recognition of a recently gained editing site. We demonstrate that changes in the RNA recognition motifs that are not explainable with the current PPR code allow an ancient PPR protein, QED1, to uniquely target the ndhB-291 site in Brassicaceae. When expressed in tobacco, the Arabidopsis QED1 edits 33 high-confident off-target sites in chloroplasts and mitochondria causing a spectrum of mutant phenotypes. By manipulating the relative expression levels of QED1 and ndhB-291, we show that the target specificity of the PPR protein depends on the RNA:protein ratio. Finally, our data suggest that the low expression levels of PPR proteins are necessary to ensure the specificity of editing site selection and prevent deleterious off-target editing.