In land plants and cyanobacteria, co-translational association of chlorophyll (Chl) to the nascent D1 polypeptide, a reaction center protein of photosystem II (PSII), requires a Chl binding complex consisting of a short-chain dehydrogenase (high chlorophyll fluorescence 244 [HCF244]/uncharacterized protein 39 [Ycf39]) and one-helix proteins (OHP1 and OHP2 in chloroplasts) of the light-harvesting antenna complex superfamily. Here, we show that an ohp2 mutant of the green alga Chlamydomonas (Chlamydomonas reinhardtii) fails to accumulate core PSII subunits, in particular D1 (encoded by the psbA mRNA). Extragenic suppressors arose at high frequency, suggesting the existence of another route for Chl association to PSII. The ohp2 mutant was complemented by the Arabidopsis (Arabidopsis thaliana) ortholog. In contrast to land plants, where psbA translation is prevented in the absence of OHP2, ribosome profiling experiments showed that the Chlamydomonas mutant translates the psbA transcript over its full length. Pulse labeling suggested that D1 is degraded during or immediately after translation. The translation of other PSII subunits was affected by assembly-controlled translational regulation. Proteomics showed that HCF244, a translation factor which associates with and is stabilized by OHP2 in land plants, still partly accumulates in the Chlamydomonas ohp2 mutant, explaining the persistence of psbA translation. Several Chl biosynthesis enzymes overaccumulate in the mutant membranes. Partial inactivation of a D1-degrading protease restored a low level of PSII activity in an ohp2 background, but not photoautotrophy. Taken together, our data suggest that OHP2 is not required for psbA translation in Chlamydomonas, but is necessary for D1 stabilization.

Abstract Photosynthesis is a central determinant of plant biomass production, but its homeostasis is increasingly challenged by heat. Little is known about the sensitive regulatory principles involved in heat acclimation that underly the biogenesis and repair of chloroplast-encoded core subunits of photosynthetic complexes. Employing time-resolved ribosome and transcript profiling together with selective ribosome proteomics, we systematically deciphered these processes in chloroplasts of Chlamydomonas reinhardtii. We revealed protein biosynthesis and altered translation elongation as central processes for heat acclimation and showed that these principles are conserved between the alga and the flowering plant Nicotiana tabacum. Short-term heat exposure resulted in specific translational repression of chlorophyll a-containing core antenna proteins of photosystems I and II. Furthermore, translocation of ribosome nascent chain complexes to thylakoid membranes was affected, as reflected by the increased accumulation of stromal cpSRP54-bound ribosomes. The successful recovery of synthesizing these proteins under prolonged acclimation of nonlethal heat conditions was associated with specific changes of the co-translational protein interaction network, including increased ribosome association of chlorophyll biogenesis enzymes and acclimation factors responsible for complex assembly. We hypothesize that co-translational cofactor binding and targeting might be bottlenecks under heat but become optimized upon heat acclimation to sustain correct co-translational protein complex assembly.

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.

Cells are highly adaptive systems that respond and adapt to changing environmental conditions such as temperature fluctuations or altered nutrient availability. Such acclimation processes involve reprogramming of the cellular gene expression profile, tuning of protein synthesis, remodeling of metabolic pathways and morphological changes of the cell shape. Nutrient starvation can lead to limited energy supply and consequently, remodeling of protein synthesis is one of the key steps of regulation since the translation of the genetic code into functional polypeptides may consume up to 40% of a cell's energy during proliferation. In eukaryotic cells, downregulation of protein synthesis during stress is mainly mediated by modification of the translation initiation factors. Prokaryotic cells suppress protein synthesis by the active formation of dimeric so-called 'hibernating' 100S ribosome complexes. Such a transition involves a number of proteins which are found in various forms in prokaryotes but also in chloroplasts of plants. Here, we review the current understanding of these hibernation factors and elaborate conserved principles which are shared between species.

Biochemical processes in chloroplasts are important for virtually all life forms. Tight regulation of protein homeostasis and the coordinated assembly of protein complexes, composed of both imported and locally synthesized subunits, are vital to plastid functionality. Protein biogenesis requires the action of cotranslationally acting molecular chaperones. One such chaperone is trigger factor (TF), which is known to cotranslationally bind most newly synthesized proteins in bacteria, thereby assisting their correct folding and maturation. However, how these processes are regulated in chloroplasts remains poorly understood. We report here functional investigation of chloroplast-localized TF (TIG1) in the green alga (Chlamydomonas reinhardtii) and the vascular land plant Arabidopsis (Arabidopsis thaliana). We show that chloroplastic TIG1 evolved as a specialized chaperone. Unlike other plastidic chaperones that are functionally interchangeable with their prokaryotic counterpart, TIG1 was not able to complement the broadly acting ortholog in Escherichia coli. Whereas general chaperone properties such as the prevention of aggregates or substrate recognition seems to be conserved between bacterial and plastidic TFs, plant TIG1s differed by associating with only a relatively small population of translating ribosomes. Furthermore, a reduction of plastidic TIG1 levels leads to deregulated protein biogenesis at the expense of increased translation, thereby disrupting the chloroplast energy household. This suggests a central role of TIG1 in protein biogenesis in the chloroplast.

Chloroplasts contain thousands of nucleus-encoded proteins that are imported from the cytosol by translocases in the chloroplast envelope membranes. Proteolytic regulation of the translocases is critically important, but little is known about the underlying mechanisms. We applied forward genetics and proteomics in Arabidopsis to identify factors required for chloroplast outer envelope membrane (OEM) protein degradation. We identified SP2, an Omp85-type β-barrel channel of the OEM, and CDC48, a cytosolic AAA+ (ATPase associated with diverse cellular activities) chaperone. Both proteins acted in the same pathway as the ubiquitin E3 ligase SP1, which regulates OEM translocase components. SP2 and CDC48 cooperated to bring about retrotranslocation of ubiquitinated substrates from the OEM (fulfilling conductance and motor functions, respectively), enabling degradation of the substrates by the 26S proteasome in the cytosol. Such chloroplast-associated protein degradation (CHLORAD) is vital for organellar functions and plant development.

Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology.

To extend our understanding of chloroplast protein import and the role played by the import machinery component Tic40, we performed a genetic screen for suppressors of chlorotic tic40 knockout mutant Arabidopsis thaliana plants. As a result, two suppressor of tic40 loci, stic1 and stic2, were identified and characterized. The stic1 locus corresponds to the gene ALBINO4 (ALB4), which encodes a paralog of the well-known thylakoid protein targeting factor ALB3. The stic2 locus identified a previously unknown stromal protein that interacts physically with both ALB4 and ALB3. Genetic studies showed that ALB4 and STIC2 act together in a common pathway that also involves cpSRP54 and cpFtsY. Thus, we conclude that ALB4 and STIC2 both participate in thylakoid protein targeting, potentially for a specific subset of thylakoidal proteins, and that this targeting pathway becomes disadvantageous to the plant in the absence of Tic40. As the stic1 and stic2 mutants both suppressed tic40 specifically (other TIC-related mutants were not suppressed), we hypothesize that Tic40 is a multifunctional protein that, in addition to its originally described role in protein import, is able to influence downstream processes leading to thylakoid biogenesis.

ALBINO3 (ALB3) is a well-known component of a thylakoid protein-targeting complex that interacts with the chloroplast signal recognition particle (cpSRP) and the cpSRP receptor, chloroplast filamentous temperature-sensitive Y (cpFtsY). Its protein-inserting function has been established mainly for light-harvesting complex proteins, which first interact with the unique chloroplast cpSRP43 component and then are delivered to the ALB3 integrase by a GTP-dependent cpSRP-cpFtsY interaction. In Arabidopsis (Arabidopsis thaliana), a subsequently discovered ALB3 homolog, ALB4, has been proposed to be involved not in light-harvesting complex protein targeting, but instead in the stabilization of the ATP synthase complex. Here, however, we show that ALB3 and ALB4 share significant functional overlap, and that both proteins are required for the efficient insertion of cytochrome f and potentially other subunits of pigment-bearing protein complexes. Genetic and physical interactions between ALB4 and ALB3, and physical interactions between ALB4 and cpSRP, suggest that the two ALB proteins may engage similar sets of interactors for their specific functions. We propose that ALB4 optimizes the insertion of thylakoid proteins by participating in the ALB3-cpSRP pathway for certain substrates (e.g. cytochrome f and the Rieske protein). Although ALB4 has clearly diverged from ALB3 in relation to the partner-recruiting C-terminal domain, our analysis suggests that one putative cpSRP-binding motif has not been entirely lost.

Plastids are a class of essential plant cell organelles comprising photosynthetic chloroplasts of green tissues, starch-storing amyloplasts of roots and tubers or the colorful pigment-storing chromoplasts of petals and fruits. They express a few genes encoded on their organellar genome, called plastome, but import most of their proteins from the cytosol. The import into plastids, the folding of freshly-translated or imported proteins, the degradation or renaturation of denatured and entangled proteins, and the quality-control of newly folded proteins all require the action of molecular chaperones. Members of all four major families of ATP-dependent molecular chaperones (chaperonin/Cpn60, Hsp70, Hsp90 and Hsp100 families) have been identified in plastids from unicellular algae to higher plants. This review aims not only at giving an overview of the most current insights into the general and conserved functions of these plastid chaperones, but also into their specific plastid functions. Given that chloroplasts harbor an extreme environment that cycles between reduced and oxidized states, that has to deal with reactive oxygen species and is highly reactive to environmental and developmental signals, it can be presumed that plastid chaperones have evolved a plethora of specific functions some of which are just about to be discovered. Here, the most urgent questions that remain unsolved are discussed, and guidance for future research on plastid chaperones is given. This article is part of a Special Issue entitled: Chloroplast Biogenesis.

Molecular chaperones play essential roles in a wide variety of cellular processes, from de-novo protein folding to protein disaggregation under stress conditions, unfolding and re-folding of misfolded proteins, protein degradation, protein transport and proteome remodeling during development. Almost all cell compartments contain chaperone activity to some extent, hence it is not surprising that a large number of chaperones also play essential roles in the plastid compartment. Here, the focus of chaperone activity is on protein targeting (protein import and assembly of complexes in target membranes) as well as protection from specific chloroplast-derived stresses. Moreover, chaperones play important roles in denovo folding of plastid-encoded proteins, in the folding of soluble proteins after import and processing of the transit peptides, and in protein degradation. The four major groups of molecular chaperones, the chaperonin/Cpn60, Hsp70, Hsp90 and Hsp100 families of chaperones, are all present in plastids but many cofactors and co-chaperones have not yet been identified. Although chaperone function is generally conserved, it seems that plastid-localized chaperones have evolved some specific functions and mechanisms. Current research on plastid-localized chaperones focuses therefore on the specificities of chaperone function in the context of their plastid environment and requirements.

Much of the chloroplast proteome is encoded in the nuclear genome and needs to be imported post-translationally. Information for the organellar targeting of these imported proteins lies in an N-terminal leader sequence, the transit peptide, which is specifically bound by receptor components at the chloroplast surface. These receptor components are part of the TOC (translocon at the outer envelope membrane of chloroplasts) complex, which, together with the TIC (translocon at the inner envelope membrane of chloroplasts) machinery, mediates the translocation of precursor proteins into chloroplasts. Apart from the receptors, these complexes incorporate channel, motor and regulatory functions. Many components of this TOC/TIC apparatus have been identified. Multiple isoforms of the TOC receptors (and possibly of some other components) enable the operation of different import pathways with different substrate preferences, perhaps so that non-abundant proteins can be imported without serious competition from highly-abundant proteins of the photosynthetic apparatus. The different import pathways might also play a role in the differentiation of different plastid types. While much research has focused on these canonical TOC/TIC-mediated import routes, a number of studies have revealed alternative protein transport pathways to chloroplasts that employ different mechanisms; one of these passes through the endoplasmic reticulum and the Golgi apparatus. Other recent studies have revealed several protein targeting pathways leading to the envelope itself.

Stromal processing peptidase (SPP) is a metalloendopeptidase located in the stroma of chloroplasts, and it is responsible for the cleavage of transit peptides from preproteins upon their import into the organelle. Two independent mutant Arabidopsis lines with T-DNA insertions in the SPP gene were analysed (spp-1 and spp-2). For both lines, no homozygous mutant plants could be detected, and the segregating progeny of spp heterozygotes contained heterozygous and wild-type plants in a ratio of 2∶1. The siliques of heterozygous spp-1 and spp-2 plants contained many aborted seeds, at a frequency of ∼25%, suggesting embryo lethality. By contrast, transmission of the spp mutations through the male and female gametes was found to be normal, and so gametophytic effects could be ruled out. To further elucidate the timing of the developmental arrest, mutant and wild-type seeds were cleared and analysed by Nomarski microscopy. A significant proportion (∼25%) of the seeds in mutant siliques exhibited delayed embryogenesis compared to those in wild type. Moreover, the mutant embryos never progressed normally beyond the 16-cell stage, with cell divisions not completing properly thereafter. Heterozygous spp mutant plants were phenotypically indistinguishable from the wild type, indicating that the spp knockout mutations are completely recessive and suggesting that one copy of the SPP gene is able to produce sufficient SPP protein for normal development under standard growth conditions.

Import of nuclear-encoded precursor proteins from the cytosol is an essential step in chloroplast biogenesis that is mediated by protein translocon complexes at the inner and outer envelope membrane (TOC). Toc159 is thought to be the main receptor for photosynthetic proteins, but lacking a large-scale systems approach, this hypothesis has only been tested for a handful of photosynthetic and nonphotosynthetic proteins. To assess Toc159 precursor specificity, we quantitatively analyzed the accumulation of plastid proteins in two mutant lines deficient in this receptor. Parallel genome-wide transcript profiling allowed us to discern the consequences of impaired protein import from systemic transcriptional responses that contribute to the loss of photosynthetic capacity. On this basis, we defined putative Toc159-independent and Toc159-dependent precursor proteins. Many photosynthetic proteins accumulate in Toc159-deficient plastids, and, surprisingly, several distinct metabolic pathways are negatively affected by Toc159 depletion. Lack of Toc159 furthermore affects several proteins that accumulate as unprocessed N-acetylated precursor proteins outside of plastids. Together, our data show an unexpected client protein promiscuity of Toc159 that requires a far more differentiated view of Toc159 receptor function and regulation of plastid protein import, in which cytosolic Met removal followed by N-terminal acetylation of precursors emerges as an additional regulatory step.

Genomic imprinting is an epigenetic phenomenon that causes monoallelic expression of specific genes dependent on the parent-of-origin. Imprinting of the Arabidopsis gene PHERES1 requires the function of the FERTILIZATION INDEPENDENT SEED (FIS) Polycomb group complex as well as a distally located methylated region containing a tandem triple repeat sequence. In this study, we investigated the regulation of the close PHERES1 homolog PHERES2. We found that PHERES2 is also a direct target gene of the FIS Polycomb group complex, but, in contrast to PHERES1, PHERES2 is equally expressed from maternal and paternal alleles. Thus, PHERES2 is not regulated by genomic imprinting, correlating with the lack of tandem repeats at PHERES2. Eliminating tandem repeats from the PHERES1 locus abolishes PHERES1 imprinting, demonstrating that tandem repeats are essential for PHERES1 imprinting. Taking these results together, our study shows that the recently duplicated genes PHERES1 and PHERES2 are both target genes of the FIS Polycomb group complex but only PHERES1 is regulated by genomic imprinting, which is likely caused by the presence of repeat sequences in the proximity of the PHERES1 locus.