A Spatial Interactome Reveals the Protein Organization of the Algal CO2-Concentrating Mechanism.

Approximately one-third of global CO2 fixation is performed by eukaryotic algae. Nearly all algae enhance their carbon assimilation by operating a CO2-concentrating mechanism (CCM) built around an organelle called the pyrenoid, whose protein composition is largely unknown. Here, we developed tools in the model alga Chlamydomonas reinhardtii to determine the localizations of 135 candidate CCM proteins and physical interactors of 38 of these proteins. Our data reveal the identity of 89 pyrenoid proteins, including Rubisco-interacting proteins, photosystem I assembly factor candidates, and inorganic carbon flux components. We identify three previously undescribed protein layers of the pyrenoid: a plate-like layer, a mesh layer, and a punctate layer. We find that the carbonic anhydrase CAH6 is in the flagella, not in the stroma that surrounds the pyrenoid as in current models. These results provide an overview of proteins operating in the eukaryotic algal CCM, a key process that drives global carbon fixation.

Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and autophagy.

UFMylation involves the covalent modification of substrate proteins with UFM1 (Ubiquitin-fold modifier 1) and is important for maintaining ER homeostasis. Stalled translation triggers the UFMylation of ER-bound ribosomes and activates C53-mediated autophagy to clear toxic polypeptides. C53 contains noncanonical shuffled ATG8-interacting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. However, the mechanistic basis of sAIM-mediated ATG8 interaction remains unknown. Here, we show that C53 and sAIMs are conserved across eukaryotes but secondarily lost in fungi and various algal lineages. Biochemical assays showed that the unicellular alga Chlamydomonas reinhardtii has a functional UFMylation pathway, refuting the assumption that UFMylation is linked to multicellularity. Comparative structural analyses revealed that both UFM1 and ATG8 bind sAIMs in C53, but in a distinct way. Conversion of sAIMs into canonical AIMs impaired binding of C53 to UFM1, while strengthening ATG8 binding. Increased ATG8 binding led to the autoactivation of the C53 pathway and sensitization of Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral role of sAIMs in UFMylation-dependent fine-tuning of C53-mediated autophagy activation.

Alternative proteoforms and proteoform-dependent assemblies in humans and plants.

The variability of proteins at the sequence level creates an enormous potential for proteome complexity. Exploring the depths and limits of this complexity is an ongoing goal in biology. Here, we systematically survey human and plant high-throughput bottom-up native proteomics data for protein truncation variants, where substantial regions of the full-length protein are missing from an observed protein product. In humans, Arabidopsis, and the green alga Chlamydomonas, approximately one percent of observed proteins show a short form, which we can assign by comparison to RNA isoforms as either likely deriving from transcript-directed processes or limited proteolysis. While some detected protein fragments align with known splice forms and protein cleavage events, multiple examples are previously undescribed, such as our observation of fibrocystin proteolysis and nuclear translocation in a green alga. We find that truncations occur almost entirely between structured protein domains, even when short forms are derived from transcript variants. Intriguingly, multiple endogenous protein truncations of phase-separating translational proteins resemble cleaved proteoforms produced by enteroviruses during infection. Some truncated proteins are also observed in both humans and plants, suggesting that they date to the last eukaryotic common ancestor. Finally, we describe novel proteoform-specific protein complexes, where the loss of a domain may accompany complex formation.

The versatile interactome of chloroplast ribosomes revealed by affinity purification mass spectrometry.

In plant cells, chloroplast gene expression is predominantly controlled through post-transcriptional regulation. Such fine-tuning is vital for precisely orchestrating protein complex assembly as for the photosynthesis machinery and for quickly responding to environmental changes. While regulation of chloroplast protein synthesis is of central importance, little is known about the degree and nature of the regulatory network, mainly due to challenges associated with the specific isolation of transient ribosome interactors. Here, we established a ribosome affinity purification method, which enabled us to broadly uncover putative ribosome-associated proteins in chloroplasts. Endogenously tagging of a protein of the large or small subunit revealed not only interactors of the holo complex, but also preferential interactors of the two subunits. This includes known canonical regulatory proteins as well as several new proteins belonging to the categories of protein and RNA regulation, photosystem biogenesis, redox control and metabolism. The sensitivity of the here applied screen was validated for various transiently interacting proteins. We further provided evidence for the existence of a ribosome-associated Nα-acetyltransferase in chloroplasts and its ability to acetylate substrate proteins at their N-terminus. The broad set of ribosome interactors underscores the potential to regulate chloroplast gene expression on the level of protein synthesis.

Coexpressed subunits of dual genetic origin define a conserved supercomplex mediating essential protein import into chloroplasts.

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.

Chloroplast proteotoxic stress-induced autophagy is involved in the degradation of chloroplast proteins in Chlamydomonas reinhardtii.

Intraorganellar proteases and cytoplasmic proteolytic systems such as autophagy orchestrate the degradation of organellar proteins to ensure organelle homeostasis in eukaryotic cells. The green alga Chlamydomonas reinhardtii is an ideal unicellular model organism for elucidating the mechanisms maintaining proteostasis in chloroplasts. However, the autophagic pathways targeting the photosynthetic organelles of these algae have not been clearly elucidated. Here, we explored the role of autophagy in chloroplast protein degradation in Chlamydomonas cells. We labeled the chloroplast protein Rubisco small subunit (RBCS) with the yellow fluorescent protein Venus in a Chlamydomonas strain in which expression of the chloroplast gene clpP1, encoding a major catalytic subunit of the chloroplast Clp protease, can be conditionally repressed to selectively perturb chloroplast protein homeostasis. We observed transport of both nucleus-encoded RBCS-Venus fusion protein and chloroplast-encoded Rubisco large subunit (rbcL) from the chloroplast to the vacuoles in response to chloroplast proteotoxic stress induced by clpP1 inhibition. This process was retarded by the addition of autophagy inhibitors. Biochemical detection of lytic cleavage of RBCS-Venus supported the notion that Rubisco is degraded in the vacuoles via autophagy. Electron microscopy revealed vacuolar accumulation of autophagic vesicles and exposed their ultrastructure during repression of clpP1 expression. Treatment with an autophagy activator also induced chloroplast autophagy. These results indicate that autophagy contributes to chloroplast protein degradation in Chlamydomonas cells.

Gordon Research Conference on photosynthesis: photosynthetic plasticity from the environment to synthetic systems.

Here, we provide a summary of the 2017 Gordon Research Conference on Photosynthesis: "Photosynthetic plasticity: from the environment to synthetic systems". This conference was held at the Grand Summit Resort Hotel at Sunday River, Newry, Maine, USA, from July 16 to 21, 2017. We have also included here a brief description of the Gordon Research Seminar (for students and post-docs) held during 2 days preceding this conference. Following the conclusion of the conference's scientific program, four young scientists (Han Bao, Vivek Tiwari, Setsuko Wakao, and Usha Lingappa) were recognized for their research presentations, each of whom received a book as a gift from one of us (Govindjee). Having chaired the 2015 Gordon Research Conference on Photosynthesis in 2015, Fabrice Rappaport, who lost his fight against cancer in January 2016, was remembered for his profound impact on the field of photosynthesis research.

Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control.

Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in environmental conditions. Proteases and molecular chaperones involved in plastid protein quality control are encoded by the nucleus except for the catalytic subunit of ClpP, an evolutionarily conserved serine protease. Unlike its Escherichia coli ortholog, this chloroplast protease is essential for cell viability. To study its function, we used a recently developed system of repressible chloroplast gene expression in the alga Chlamydomonas reinhardtii. Using this repressible system, we have shown that a selective gradual depletion of ClpP leads to alteration of chloroplast morphology, causes formation of vesicles, and induces extensive cytoplasmic vacuolization that is reminiscent of autophagy. Analysis of the transcriptome and proteome during ClpP depletion revealed a set of proteins that are more abundant at the protein level, but not at the RNA level. These proteins may comprise some of the ClpP substrates. Moreover, the specific increase in accumulation, both at the RNA and protein level, of small heat shock proteins, chaperones, proteases, and proteins involved in thylakoid maintenance upon perturbation of plastid protein homeostasis suggests the existence of a chloroplast-to-nucleus signaling pathway involved in organelle quality control. We suggest that this represents a chloroplast unfolded protein response that is conceptually similar to that observed in the endoplasmic reticulum and in mitochondria.

Conditional repression of essential chloroplast genes: Evidence for new plastid signaling pathways.

The development of a repressible chloroplast gene expression system in Chlamydomonas reinhardtii has opened the door for studying the role of essential chloroplast genes. This approach has been used to analyze three chloroplast genes of this sort coding for the α subunit of RNA polymerase (rpoA), a ribosomal protein (rps12) and the catalytic subunit of the ATP-dependent ClpP protease (clpP1). Depletion of the three corresponding proteins leads to growth arrest and cell death. Shutdown of chloroplast transcription and translation increases the abundance of a set of plastid transcripts that includes mainly those involved in transcription, translation and proteolysis and reveals multiple regulatory feedback loops in the chloroplast gene circuitry. Depletion of ClpP profoundly affects plastid protein homeostasis and elicits an autophagy-like response with extensive cytoplasmic vacuolization of cells. It also triggers changes in chloroplast and nuclear gene expression resulting in increased abundance of chaperones, proteases, ubiquitin-related proteins and proteins involved in lipid trafficking and thylakoid biogenesis. These features are hallmarks of an unfolded protein response in the chloroplast and raise new questions on plastid protein homeostasis and plastid signaling. This article is part of a Special Issue entitled: Chloroplast Biogenesis.

Repression of essential chloroplast genes reveals new signaling pathways and regulatory feedback loops in chlamydomonas.

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.

Repressible chloroplast gene expression in Chlamydomonas: a new tool for the study of the photosynthetic apparatus.

A repressible/inducible chloroplast gene expression system has been used to conditionally inhibit chloroplast protein synthesis in the unicellular alga Chlamydomonas reinhardtii. This system allows one to follow the fate of photosystem II and photosystem I and their antennae upon cessation of chloroplast translation. The main results are that the levels of the PSI core proteins decrease at a slower rate than those of PSII. Amongst the light-harvesting complexes, the decrease of CP26 proceeds at the same rate as for the PSII core proteins whereas it is significantly slower for CP29, and for the antenna complexes of PSI this rate is comprised between that of CP26 and CP29. In marked contrast, the components of trimeric LHCII, the major PSII antenna, persist for several days upon inhibition of chloroplast translation. This system offers new possibilities for investigating the biosynthesis and turnover of individual photosynthetic complexes in the thylakoid membranes. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

Systematic characterization of gene function in the photosynthetic alga Chlamydomonas reinhardtii.

Most genes in photosynthetic organisms remain functionally uncharacterized. Here, using a barcoded mutant library of the model eukaryotic alga Chlamydomonas reinhardtii, we determined the phenotypes of more than 58,000 mutants under more than 121 different environmental growth conditions and chemical treatments. A total of 59% of genes are represented by at least one mutant that showed a phenotype, providing clues to the functions of thousands of genes. Mutant phenotypic profiles place uncharacterized genes into functional pathways such as DNA repair, photosynthesis, the CO2-concentrating mechanism and ciliogenesis. We illustrate the value of this resource by validating phenotypes and gene functions, including three new components of an actin cytoskeleton defense pathway. The data also inform phenotype discovery in land plants; mutants in Arabidopsis thaliana genes exhibit phenotypes similar to those we observed in their Chlamydomonas homologs. We anticipate that this resource will guide the functional characterization of genes across the tree of life.

Regulated Chloroplast Gene Expression in Chlamydomonas.

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 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.

The Mars1 kinase confers photoprotection through signaling in the chloroplast unfolded protein response.

In response to proteotoxic stress, chloroplasts communicate with the nuclear gene expression system through a chloroplast unfolded protein response (cpUPR). We isolated Chlamydomonas reinhardtii mutants that disrupt cpUPR signaling and identified a gene encoding a previously uncharacterized cytoplasmic protein kinase, termed Mars1-for mutant affected in chloroplast-to-nucleus retrograde signaling-as the first known component in cpUPR signal transmission. Lack of cpUPR induction in MARS1 mutant cells impaired their ability to cope with chloroplast stress, including exposure to excessive light. Conversely, transgenic activation of cpUPR signaling conferred an advantage to cells undergoing photooxidative stress. Our results indicate that the cpUPR mitigates chloroplast photodamage and that manipulation of this pathway is a potential avenue for engineering photosynthetic organisms with increased tolerance to chloroplast stress.

Life on Earth crucially depends on photosynthesis, the process by which energy stored in sunlight is harnessed to convert carbon dioxide into sugars and oxygen. In plants and algae, photosynthesis occurs in specialized cellular compartments called chloroplasts. Inside chloroplasts, complex molecular machines absorb light and channel its energy into the appropriate chemical reactions. These machines are composed of proteins that need to be assembled and maintained. However, proteins can become damaged, and when this occurs, they must be recognized, removed, and replaced. When exposed to bright light, the photosynthetic machinery is pushed into overdrive and protein damage is accelerated. In response, the chloroplast sends an alarm signal to activate a protective system called the "chloroplast unfolded protein response", or cpUPR for short. The cpUPR leads to the production of specialized proteins that help protect and repair the chloroplast. It was not known how plants and algae evaluate the level of damaged proteins in the chloroplast, or which signals trigger the cpUPR. To address these questions, Perlaza et al. designed a method to identify the molecular components of the alarm signal. These experiments used specially engineered cells from the algae Chlamydomonas reinhardtii that fluoresced when the cpUPR was activated. Perlaza et al. mutagenized these cells ­ that is, damaged the cells' DNA to cause random changes in the genetic code. If a mutagenized cell no longer fluoresced in response to protein damage, it indicated that communication between protein damage and the cpUPR had been broken. In other words, the mutation had damaged a piece of DNA that encoded a protein critical for activating the cpUPR. These experiments identified one protein ­ which Perlaza et al. named Mars1 ­ as a crucial molecular player that is required to trigger the cpUPR. Algal cells with defective Mars1 were more vulnerable to chloroplast damage, including that caused by excessive light. These discoveries in algae will serve as a foundation for understanding the mechanism and significance of the cpUPR in land plants. Perlaza et al. also found that mild artificial activation of the cpUPR could preemptively guard cells against damaged chloroplast proteins. This suggests that the cpUPR could be harnessed in agriculture, for example, to help crop plants endure harsher climates.

A genome-wide algal mutant library and functional screen identifies genes required for eukaryotic photosynthesis.

Photosynthetic organisms provide food and energy for nearly all life on Earth, yet half of their protein-coding genes remain uncharacterized1,2. Characterization of these genes could be greatly accelerated by new genetic resources for unicellular organisms. Here we generated a genome-wide, indexed library of mapped insertion mutants for the unicellular alga Chlamydomonas reinhardtii. The 62,389 mutants in the library, covering 83% of nuclear protein-coding genes, are available to the community. Each mutant contains unique DNA barcodes, allowing the collection to be screened as a pool. We performed a genome-wide survey of genes required for photosynthesis, which identified 303 candidate genes. Characterization of one of these genes, the conserved predicted phosphatase-encoding gene CPL3, showed that it is important for accumulation of multiple photosynthetic protein complexes. Notably, 21 of the 43 higher-confidence genes are novel, opening new opportunities for advances in understanding of this biogeochemically fundamental process. This library will accelerate the characterization of thousands of genes in algae, plants, and animals.

Chloroplast signaling and quality control.

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.

Controlling expression of genes in the unicellular alga Chlamydomonas reinhardtii with a vitamin-repressible riboswitch.

Chloroplast genomes of land plants and algae contain generally between 100 and 150 genes. These genes are involved in plastid gene expression and photosynthesis and in various other tasks. The function of some chloroplast genes is still unknown and some of them appear to be essential for growth and survival. Repressible and reversible expression systems are highly desirable for functional and biochemical characterization of these genes. We have developed a genetic tool that allows one to regulate the expression of any coding sequence in the chloroplast genome of the unicellular alga Chlamydomonas reinhardtii. Our system is based on vitamin-regulated expression of the nucleus-encoded chloroplast Nac2 protein, which is specifically required for the expression of any plastid gene fused to the psbD 5'UTR. With this approach, expression of the Nac2 gene in the nucleus and, in turn, that of the chosen chloroplast gene artificially driven by the psbD 5'UTR, is controlled by the MetE promoter and Thi4 riboswitch, which can be inactivated in a reversible way by supplying vitamin B12 and thiamine to the growth medium, respectively. This system opens interesting possibilities for studying the assembly and turnover of chloroplast multiprotein complexes such as the photosystems, the ribosome, and the RNA polymerase. It also provides a way to overcome the toxicity often associated with the expression of proteins of biotechnological interest in the chloroplast.

Chloroplast unfolded protein response, a new plastid stress signaling pathway?

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.

Loss of chloroplast ClpP elicits an autophagy-like response in Chlamydomonas.

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.

Tools for regulated gene expression in the chloroplast of Chlamydomonas.

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.