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Chloroplasts rely on the translocon complexes in the outer and inner envelope membranes (the TOC and TIC complexes, respectively) to import thousands of different nuclear-encoded proteins from the cytosol 1-4 . Although previous studies indicated that the TOC and TIC complexes may assemble into larger supercomplexes 5-7 , the overall architectures of the TOC-TIC supercomplexes and the mechanism of preprotein translocation are unclear. Here we report the cryo-electron microscopy structure of the TOC-TIC supercomplex from Chlamydomonas reinhardtii. The major subunits of the TOC complex (Toc75, Toc90 and Toc34) and TIC complex (Tic214, Tic20, Tic100 and Tic56), three chloroplast translocon-associated proteins (Ctap3, Ctap4 and Ctap5) and three newly identified small inner-membrane proteins (Simp1-3) have been located in the supercomplex. As the largest protein, Tic214 traverses the inner membrane, the intermembrane space and the outer membrane, connecting the TOC complex with the TIC proteins. An inositol hexaphosphate molecule is located at the Tic214-Toc90 interface and stabilizes their assembly. Four lipid molecules are located within or above an inner-membrane funnel formed by Tic214, Tic20, Simp1 and Ctap5. Multiple potential pathways found in the TOC-TIC supercomplex may support translocation of different substrate preproteins into chloroplasts., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Most chloroplast proteins are nucleus-encoded, translated on cytoplasmic ribosomes as precursor proteins, and imported into chloroplasts through TOC and TIC, the translocons of the outer and inner chloroplast envelope membranes. While the composition of the TOC complex is well established, there is still some controversy about the importance of a recently identified TIC complex consisting of Tic20, Tic214, Tic100, and Tic56. TOC and TIC form a supercomplex with a protein channel at the junction of the outer and inner envelope membranes through which preproteins are pulled into the stroma by the ATP-powered Ycf2 complex consisting of several FtsH-like ATPases and/or by chloroplast Hsp proteins. Several components of the TOC/TIC system are moonlighting proteins with additional roles in chloroplast gene expression and metabolism. Chaperones and co-chaperones, associated with TOC and TIC on the cytoplasmic and stromal side of the chloroplast envelope, participate in the unfolding and folding of the precursor proteins and act together with the ubiquitin-proteasome system in protein quality control. Chloroplast protein import is also intimately linked with retrograde signaling, revealing altogether an unsuspected complexity in the regulation of this process., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Photosynthesis and the biosynthesis of many important metabolites occur in chloroplasts. In these semi-autonomous organelles, the chloroplast genome encodes approximately 100 proteins. The remaining chloroplast proteins, close to 3,000, are encoded by nuclear genes whose products are translated in the cytosol and imported into chloroplasts. However, there is still no consensus on the composition of the protein import machinery including its motor proteins and on how newly imported chloroplast proteins are refolded. In this study, we have examined the function of orf2971, the largest chloroplast gene of Chlamydomonas reinhardtii. The depletion of Orf2971 causes the accumulation of protein precursors, partial proteolysis and aggregation of proteins, increased expression of chaperones and proteases, and autophagy. Orf2971 interacts with the TIC (translocon at the inner chloroplast envelope) complex, catalyzes ATP (adenosine triphosphate) hydrolysis, and associates with chaperones and chaperonins. We propose that Orf2971 is intimately connected to the protein import machinery and plays an important role in chloroplast protein quality control., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Fe-S clusters are ancient, ubiquitous and highly essential prosthetic groups for numerous fundamental processes of life. The biogenesis of Fe-S clusters is a multistep process including iron acquisition, sulfur mobilization, and cluster formation. Extensive studies have provided deep insights into the mechanism of the latter two assembly steps. However, the mechanism of iron utilization during chloroplast Fe-S cluster biogenesis is still unknown. Here we identified two Arabidopsis DnaJ proteins, DJA6 and DJA5, that can bind iron through their conserved cysteine residues and facilitate iron incorporation into Fe-S clusters by interactions with the SUF (sulfur utilization factor) apparatus through their J domain. Loss of these two proteins causes severe defects in the accumulation of chloroplast Fe-S proteins, a dysfunction of photosynthesis, and a significant intracellular iron overload. Evolutionary analyses revealed that DJA6 and DJA5 are highly conserved in photosynthetic organisms ranging from cyanobacteria to higher plants and share a strong evolutionary relationship with SUFE1, SUFC, and SUFD throughout the green lineage. Thus, our work uncovers a conserved mechanism of iron utilization for chloroplast Fe-S cluster biogenesis., (© 2021 The Authors.)

The green unicellular alga Chlamydomonas reinhardtii has emerged as a very attractive model system for chloroplast genetic engineering. Algae can be transformed readily at the chloroplast level through bombardment of cells with a gene gun and transformants can be selected using antibiotic resistance or phototrophic growth. An inducible chloroplast gene expression system could be very useful for several reasons. First, it could be used to elucidate the function of essential chloroplast genes required for cell growth and survival. Second, it could be very helpful for expressing proteins which are toxic to the algal cells. Third, it would allow for the reversible depletion of photosynthetic complexes, thus making it possible to study their biogenesis in a controlled fashion. Fourth, it opens promising possibilities for hydrogen production in Chlamydomonas. Here we describe an inducible/ repressible chloroplast gene expression system in Chlamydomonas in which the copper-regulated Cyc 6 promoter or the vitamin-controlled MetE promoter and TPP riboswitch drive the expression of the nuclear Nac2 gene encoding a protein which is targeted to the chloroplast where it acts specifically on the chloroplast psbD 5' untranslated region and is required for the stable accumulation of the psbD mRNA and photosystem II. The system can be used for any chloroplast gene or trans-gene by placing it under the control of the psbD 5'untranslated region.

In photosynthetic eukaryotes, thousands of proteins are translated in the cytosol and imported into the chloroplast through the concerted action of two translocons-termed TOC and TIC-located in the outer and inner membranes of the chloroplast envelope, respectively. The degree to which the molecular composition of the TOC and TIC complexes is conserved over phylogenetic distances has remained controversial. Here, we combine transcriptomic, biochemical, and genetic tools in the green alga Chlamydomonas ( Chlamydomonas reinhardtii ) to demonstrate that, despite a lack of evident sequence conservation for some of its components, the algal TIC complex mirrors the molecular composition of a TIC complex from Arabidopsis thaliana. The Chlamydomonas TIC complex contains three nuclear-encoded subunits, Tic20, Tic56, and Tic100, and one chloroplast-encoded subunit, Tic214, and interacts with the TOC complex, as well as with several uncharacterized proteins to form a stable supercomplex (TIC-TOC), indicating that protein import across both envelope membranes is mechanistically coupled. Expression of the nuclear and chloroplast genes encoding both known and uncharacterized TIC-TOC components is highly coordinated, suggesting that a mechanism for regulating its biogenesis across compartmental boundaries must exist. Conditional repression of Tic214, the only chloroplast-encoded subunit in the TIC-TOC complex, impairs the import of chloroplast proteins with essential roles in chloroplast ribosome biogenesis and protein folding and induces a pleiotropic stress response, including several proteins involved in the chloroplast unfolded protein response. These findings underscore the functional importance of the TIC-TOC supercomplex in maintaining chloroplast proteostasis., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Members of the mitochondrial transcription terminator factor (mTERF) family, originally identified in vertebrate mitochondria, are involved in the termination of organellular transcription. In plants, mTERF proteins are mainly localized in chloroplasts and mitochondria. In Arabidopsis ( Arabidopsis thaliana ), mTERF8/pTAC15 was identified in the plastid-encoded RNA polymerase (PEP) complex, the major RNA polymerase of chloroplasts. In this work, we demonstrate that mTERF8 is associated with the PEP complex. An mTERF8 knockout line displayed a wild-type-like phenotype under standard growth conditions, but showed impaired efficiency of photosystem II electron flow. Transcription of most chloroplast genes was not substantially affected in the mterf8 mutant; however, the level of the psbJ transcript from the psbEFLJ polycistron was increased. RNA blot analysis showed that a larger transcript accumulates in mterf8 than in the wild type. Thus, abnormal transcription and/or RNA processing occur for the psbEFLJ polycistron. Circular reverse transcription PCR and sequence analysis showed that the psbJ transcript terminates 95 nucleotides downstream of the translation stop codon in the wild type, whereas its termination is aberrant in mterf8 Both electrophoresis mobility shift assays and chloroplast chromatin immunoprecipitation analysis showed that mTERF8 specifically binds to the 3' terminal region of psbJ Transcription analysis using the in vitro T7 RNA polymerase system showed that mTERF8 terminates psbJ transcription. Together, these results suggest that mTERF8 is specifically involved in the transcription termination of the chloroplast gene psbJ ., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

F-type ATP synthases produce nearly all of the ATP found in cells. The catalytic module F 1 commonly comprises an α 3 β 3 hexamer surrounding a γ/ε stalk. However, it is unclear how these subunits assemble to form a catalytic motor. In this work, we identified and characterized a chloroplast protein that interacts with the CF 1 β, γ, and ε subunits of the chloroplast ATP synthase and is required for assembly of its F 1 module. We named this protein BIOGENESIS FACTOR REQUIRED FOR ATP SYNTHASE1 (BFA1) and determined its crystal structure at 2.8-Å resolution. BFA1 is comprised primarily of two interacting β-barrels that are oriented nearly perpendicularly to each other. The contact region between BFA1 and the CF 1 β and γ subunits was further mapped by yeast two-hybrid assays. An in silico molecular docking analysis was performed and revealed close fitting contact sites without steric conflicts between BFA1 and CF 1 β/γ. We propose that BFA1 acts mainly as a scaffold protein promoting the association of a CF 1 α/β heterodimer with CF 1 γ. The subsequent assembly of other CF 1 α/β heterodimers may shift the position of the CF 1 γ subunit to complete assembly of the CF 1 module. This CF 1 assembly process is likely to be valid for other F-type ATP synthases, as their structures are highly conserved., (© 2018 American Society of Plant Biologists. All rights reserved.)

Singlet oxygen ( 1 O 2 ) is a prime cause of photo-damage of the photosynthetic apparatus. The chlorophyll molecules in the photosystem II reaction center and in the light-harvesting antenna complex are major sources of 1 O 2 generation. It has been thought that the generation of 1 O 2 mainly takes place in the appressed regions of the thylakoid membranes, namely, the grana core, where most of the active photosystem II complexes are localized. Apart from being a toxic molecule, new evidence suggests that 1 O 2 significantly contributes to chloroplast-to-nucleus retrograde signalling that primes acclimation and cell death responses. Interestingly, recent studies reveal that chloroplasts operate two distinct 1 O 2 -triggered retrograde signalling pathways in which β-carotene and a nuclear-encoded chloroplast protein EXECUTER1 play essential roles as signalling mediators. The coexistence of these mediators raises several questions: their crosstalk, source(s) of 1 O 2 , downstream signalling components, and the perception and reaction mechanism of these mediators towards 1 O 2 . In this review, we mainly discuss the molecular genetic basis of the mode of action of these two putative 1 O 2 sensors and their corresponding retrograde signalling pathways. In addition, we also propose the possible existence of an alternative source of 1 O 2 , which is spatially and functionally separated from the grana core., (© 2018 John Wiley & Sons Ltd.)

Although chloroplasts contain their own genetic system and are semi-autonomous cell organelles, plastid biogenesis and homeostasis are heavily dependent on the nucleo-cytosolic compartment. These two cellular compartments are closely co-ordinated through a complex signaling network comprising both anterograde and retrograde signaling chains. Developmental changes or any perturbation in the chloroplast system induced by a particular stress resulting from changes in environmental conditions such as excess light, elevated temperature, nutrient limitation, pathogen infection, give rise to specific signals. They migrate out of the chloroplast and are perceived by the nucleus where they elicit changes in expression of particular genes that allow for the maintenance of plastid homeostasis toward environmental cues. These genes mainly include those of photosynthesis-associated proteins, chaperones, proteases, nucleases and immune/defense proteins. Besides this transcriptional response, a chloroplast quality control system exists that is involved in the repair and turnover of damaged plastid proteins. This system degrades aggregated or damaged proteins and it can even remove entire chloroplasts when they have suffered heavy damage. This response comprises several processes such as plastid autophagy and ubiquitin-proteasome mediated proteolysis that occurs on the plastid envelope through the action of the ubiquitin-proteasome system., (© 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The pyrenoid is a membrane-less organelle that exists in various photosynthetic organisms, such as algae, and wherein most global CO 2 fixation occurs. Two papers from the Jonikas lab in this issue of Cell provide new insights into the structure, protein composition, and dynamics of this important organelle., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Chloroplast genomes contain a single ClpP1 gene encoding one of the catalytic subunits of the evolutionarily conserved ATP-dependent Clp protease. Efforts to inactivate this protease in the chloroplast through targeted disruption of the clpP1 gene have failed, suggesting that it is essential for cell survival in plants. To circumvent this problem, a repressible chloroplast gene expression system was developed in the green unicellular alga Chlamydomonas reinhardtii. This system takes advantage of the nuclear Nac2 gene fused to the MetE promoter and Thi4 riboswitch, which can be repressed by adding vitamin B12 and thiamine to the growth medium. Nac2 encodes a chloroplast protein that interacts specifically with the 5'UTR of the psbD mRNA and is involved in processing/translation of this transcript. Loss of Nac2 leads to the specific degradation of psbD mRNA. Because the psbD 5'UTR is necessary and sufficient for the Nac2-dependent stability of psbD mRNA, this dependence can be transferred to any chloroplast gene by linking its coding sequence to the psbD 5 'UTR. In this way it was possible to repress the clpP1 gene in a reversible way with vitamins.

A unique feature of the ATP-dependent ClpP protease of eukaryotic photosynthetic organisms is that its catalytic subunit ClpP1 is encoded by the chloroplast genome. Attempts to inactivate this subunit through chloroplast transformation have failed because it is essential for cell survival. To study the function of ClpP we have developed a repressible chloroplast gene expression system in Chlamydomonas reinhardtii. This system is based on the use of a chimeric nuclear gene in which the vitamin-repressible MetE promoter and Thi4 riboswitch have been fused to the coding sequence of Nac2. Upon entry into the chloroplast the Nac2 protein specifically interacts with the psbD 5'UTR and is required for the proper processing/translation of the psbD mRNA. This property can be conveyed to any chloroplast mRNA by replacing its 5'UTR with that of psbD. In this study we have chosen clpP1 as plastid target gene and examined the cellular events induced upon depletion of ClpP through transcriptomic, proteomic, biochemical and electron microscope analysis. Among the most striking features, a massive increase in protein abundance occurs for plastid chaperones, proteases and proteins involved in membrane assembly/disassembly strongly suggesting the existence of a chloroplast unfolded protein response.

The green unicellular alga Chlamydomonas reinhardtii has emerged as a very attractive model system for chloroplast genetic engineering. Algae can be transformed readily at the chloroplast level through bombardment of cells with a gene gun, and transformants can be selected using antibiotic resistance or phototrophic growth. An inducible chloroplast gene expression system could be very useful for several reasons. First, it could be used to elucidate the function of essential chloroplast genes required for cell growth and survival. Second, it could be very helpful for expressing proteins which are toxic to the algal cells. Third, it would allow for the reversible depletion of photosynthetic complexes thus making it possible to study their biogenesis in a controlled fashion. Fourth, it opens promising possibilities for hydrogen production in Chlamydomonas. Here we describe an inducible/repressible chloroplast gene expression system in Chlamydomonas in which the copper-regulated Cyc6 promoter drives the expression of the nuclear Nac2 gene encoding a protein which is targeted to the chloroplast where it acts specifically on the chloroplast psbD 5'-untranslated region and is required for the stable accumulation of the psbD mRNA and photosystem II. The system can be used for any chloroplast gene or transgene by placing it under the control of the psbD 5'-untranslated region.

Although reverse genetics has been used to elucidate the function of numerous chloroplast proteins, the characterization of essential plastid genes and their role in chloroplast biogenesis and cell survival has not yet been achieved. Therefore, we developed a robust repressible chloroplast gene expression system in the unicellular alga Chlamydomonas reinhardtii based mainly on a vitamin-repressible riboswitch, and we used this system to study the role of two essential chloroplast genes: ribosomal protein S12 (rps12), encoding a plastid ribosomal protein, and rpoA, encoding the α-subunit of chloroplast bacterial-like RNA polymerase. Repression of either of these two genes leads to the arrest of cell growth, and it induces a response that involves changes in expression of nuclear genes implicated in chloroplast biogenesis, protein turnover, and stress. This response also leads to the overaccumulation of several plastid transcripts and reveals the existence of multiple negative regulatory feedback loops in the chloroplast gene circuitry.

Chloroplast transformation in microalgae offers great promise for the production of proteins of pharmaceutical interest or for the development of novel biofuels. For many applications, high level expression of transgenes is desirable. We have transformed the chloroplast of Chlamydomonas reinhardtii with two genes, acrV and vapA, which encode antigens from the fish pathogen Aeromonas salmonicida. The promoters and 5' untranslated regions of four chloroplast genes were compared for their ability to drive expression of the bacterial genes. The highest levels of expression were obtained when they were placed under the control of the cis-acting elements from the psaA-exon1 gene. The expression of these chimeric genes was further increased when a nuclear mutation that affects a factor involved in psaA splicing was introduced in the genetic background of the chloroplast transformants. Accumulation of both the chimeric mRNAs and the recombinant proteins was dramatically increased, indicating that negative feedback loops limit the expression of chloroplast transgenes. Our results demonstrate the potential of manipulating anterograde signalling to alter negative regulatory feedback loops in the chloroplast and improve transgene expression., (© 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.)

An inducible chloroplast gene expression system was developed in Chlamydomonas reinhardtii by taking advantage of the properties of the copper-sensitive cytochrome c(6) promoter and of the nucleus-encoded Nac2 chloroplast protein. This protein is specifically required for the stable accumulation of the chloroplast psbD RNA and acts on its 5' UTR. A construct containing the Nac2 coding sequence fused to the cytochrome c(6) promoter was introduced into the nac2-26 mutant strain deficient in Nac2. In this transformant, psbD is expressed in copper-depleted but not in copper-replete medium. Because psbD encodes the D2 reaction center polypeptide of photosystem II (PSII), the repression of psbD leads to the loss of PSII. We have tested this system for hydrogen production. Upon addition of copper to cells pregrown in copper-deficient medium, PSII levels declined to a level at which oxygen consumption by respiration exceeded oxygen evolution by PSII. The resulting anaerobic conditions led to the induction of hydrogenase activity. Because the Cyc6 promoter is also induced under anaerobic conditions, this system opens possibilities for sustained cycling hydrogen production. Moreover, this inducible gene expression system is applicable to any chloroplast gene by replacing its 5' UTR with the psbD 5' UTR in the same genetic background. To make these strains phototrophic, the 5' UTR of the psbD gene was replaced by the petA 5' UTR. As an example, we show that the reporter gene aadA driven by the psbD 5' UTR confers resistance to spectinomycin in the absence of copper and sensitivity in its presence in the culture medium.

In the chloroplast of Chlamydomonas reinhardtii, psaA mRNA is spliced in trans from three separate precursors which assemble to form two group II introns. A fourth transcript, tscA, completes the structure of the first intron. Of the fourteen nucleus-encoded factors involved in psaA splicing, only two are required for splicing of both introns. We cloned and characterized the first of these more general factors, Raa1. Consistently with its role in psaA splicing, Raa1 is imported in the chloroplast where it is found in a membrane fraction and is part of a large ribonucleoprotein complex. One mutant, raa1-L137H, is defective for splicing of both introns, but another allelic mutant, raa1-314B, still expresses the 3' part of the Raa1 gene and is deficient only in splicing of intron 2. This observation and a deletion analysis indicate the presence of two domains in Raa1. The C-terminal domain is necessary and sufficient for processing of tscA RNA and splicing of the first intron, while the central domain is essential for splicing of the second intron. The combination of these two functional domains in Raa1 suggests that this new factor may coordinate trans-splicing of the two introns to improve the efficiency of psaA maturation.

The psaA gene of the green alga Chlamydomonas reinhardtii consists of three exons that are widely separated on the chloroplast genome and transcribed independently. The exons are flanked by group II intron sequences. Maturation of the psaA mRNA requires two steps of splicing in trans between the transcripts of exons 1, 2, and 3. At least 14 nuclear loci and one chloroplast gene (tscA) are involved in this process. Recently the genes of three of these nuclear factors have been cloned. Raa3 is involved in the first trans-splicing reaction, and Raa1 and Raa2 are required for the second trans-splicing reaction. Here we show that Raa1 and Raa2 can be coimmunoprecipitated and that they are part of a high molecular weight complex of 400-500 kD. The size and integrity of the complex are affected by mutations in other complementation groups, suggesting that the corresponding proteins may also be components of this multiprotein complex or required for its assembly. Raa1 is also associated with a larger complex.

The chloroplast psaB mRNA encodes one of the reaction centre polypeptides of photosystem I. Protein pulse-labelling profiles indicate that the mutant strain of Chlamydomonas reinhardtii, F14, affected at the nuclear locus TAB2, is deficient in the translation of psaB mRNA and thus deficient in photosystem I activity. Genetic studies reveal that the target site for Tab2 is situated within the psaB 5'UTR. We have used genomic complementation to isolate the nuclear Tab2 gene. The deduced amino acid sequence of Tab2 (358 residues) displays 31-46% sequence identity with several orthologues found only in eukaryotic and prokaryotic organisms performing oxygenic photosynthesis. Directed mutagenesis indicates the importance of a highly conserved C-terminal tripeptide in Tab2 for normal psaB translation. The Tab2 protein is localized in the chloroplast stroma where it is associated with a high molecular mass protein complex containing the psaB mRNA. Gel mobility shift assays reveal a direct and specific interaction between Tab2 and the psaB 5'UTR. We propose that Tab2 plays a key role in the initial steps of PsaB translation and photosystem I assembly.

The gene encoding the chloroplast ribosomal protein S1 from Chlamydomonas reinhardtii, CreS1, was cloned and the RNA binding properties and the expression patterns were studied. Gel-shift analysis revealed that CreS1 binds AU-rich 5'-untranslated regions (5'-UTR) of chloroplast mRNAs with higher affinity than the corresponding sequence of a GC-rich nuclear transcript. The binding affinity of CreS1 for a mutant form of the psbD 5'-UTR with a deletion of a U-rich stretch that is required for translation decreases 4-fold as compared to the wild-type 5'-UTR. Our results suggest that CreS1 protein interacts with U-rich sequences. Most of CreS1 is bound to high-molecular-weight complexes which co-migrate with the 30S small ribosomal subunit, and only a small fraction of CreS1 exists in its free form. CreS1 is localized mainly to the chloroplast stroma albeit a significant fraction is associated with chloroplast membranes. The results suggest that most of CreS1 is associated with the 30S ribosomal subunit throughout the translation process. Upon a shift of cells from the dark to the light, the mRNA levels of CreS1 and Psrp-7, both components of the 30S ribosomal subunit, increase transiently and return to the dark levels after 8 h. However, during this dark-to-light transition the levels of CreS1 and of other components of the 30S subunit remain the same suggesting that either protein synthesis or degradation is regulated. The possible implications of these findings are discussed.

Translation of the chloroplast psbC mRNA in the unicellular eukaryotic alga Chlamydomonas reinhardtii is controlled by interactions between its 547-base 5' untranslated region and the products of the nuclear loci TBC1, TBC2, and possibly TBC3. In this study, a series of site-directed mutations in this region was generated and the ability of these constructs to drive expression of a reporter gene was assayed in chloroplast transformants that are wild type or mutant at these nuclear loci. Two regions located in the middle of the 5' leader and near the initiation codon are important for translation. Other deletions still allow for partial expression of the reporter gene in the wild-type background. Regions with target sites for TBC1 and TBC2 were identified by estimating the residual translation activity in the respective mutant backgrounds. TBC1 targets include mostly the central part of the leader and the translation initiation region whereas the only detected TBC2 targets are in the 3' part. The 5'-most 93 nt of the leader are required for wild-type levels of transcription and/or mRNA stabilization. The results indicate that TBC1 and TBC2 function independently and further support the possibility that TBC1 acts together with TBC3.

Summary: R‐loops, three‐stranded nucleic acid structures consisting of a DNA: RNA hybrid and displaced single‐stranded DNA, play critical roles in gene expression and genome stability. How R‐loop homeostasis is integrated into chloroplast gene expression remains largely unknown.We found an unexpected function of FtsHi1, an inner envelope membrane‐bound AAA‐ATPase in chloroplast R‐loop homeostasis of Arabidopsis thaliana.Previously, this protein was shown to function as a component of the import motor complex for nuclear‐encoded chloroplast proteins. However, this study provides evidence that FtsHi1 is an ATP‐dependent helicase that efficiently unwinds both DNA–DNA and DNA–RNA duplexes, thereby preventing R‐loop accumulation. Over‐accumulation of R‐loops could impair chloroplast transcription but not necessarily genome integrity. The dual function of FtsHi1 in both protein import and chloroplast gene expression may be important to coordinate the biogenesis of nuclear‐ and chloroplast‐encoded subunits of multi‐protein photosynthetic complexes.This study suggests a mechanical link between protein import and R‐loop homeostasis in chloroplasts of higher plants. [ABSTRACT FROM AUTHOR]