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.

Removal of the large inverted repeat from the plastid genome reveals gene dosage effects and leads to increased genome copy number.

The chloroplast genomes of most plants and algae contain a large inverted repeat (IR) region that separates two single-copy regions and harbours the ribosomal RNA operon. We have addressed the functional importance of the IR region by removing an entire copy of the 25.3-kb IR from the tobacco plastid genome. Using plastid transformation and subsequent selectable marker gene elimination, we precisely excised the IR, thus generating plants with a substantially reduced plastid genome size. We show that the lack of the IR results in a mildly reduced plastid ribosome number, suggesting a gene dosage benefit from the duplicated presence of the ribosomal RNA operon. Moreover, the IR deletion plants contain an increased number of plastid genomes, suggesting that genome copy number is regulated by measuring total plastid DNA content rather than by counting genomes. Together, our findings (1) demonstrate that the IR can enhance the translation capacity of the plastid, (2) reveal the relationship between genome size and genome copy number, and (3) provide a simplified plastid genome structure that will facilitate future synthetic biology applications.

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.

Growth in fluctuating light buffers plants against photorespiratory perturbations.

Photorespiration (PR) is the pathway that detoxifies the product of the oxygenation reaction of Rubisco. It has been hypothesized that in dynamic light environments, PR provides a photoprotective function. To test this hypothesis, we characterized plants with varying PR enzyme activities under fluctuating and non-fluctuating light conditions. Contrasting our expectations, growth of mutants with decreased PR enzyme levels was least affected in fluctuating light compared with wild type. Results for growth, photosynthesis and metabolites combined with thermodynamics-based flux analysis revealed two main causal factors for this unanticipated finding: reduced rates of photosynthesis in fluctuating light and complex re-routing of metabolic fluxes. Only in non-fluctuating light, mutants lacking the glutamate:glyoxylate aminotransferase 1 re-routed glycolate processing to the chloroplast, resulting in photooxidative damage through H2O2 production. Our results reveal that dynamic light environments buffer plant growth and metabolism against photorespiratory perturbations.

Targeted knockout of a conserved plant mitochondrial gene by genome editing.

Fusion proteins derived from transcription activator-like effectors (TALEs) have emerged as genome editing tools for mitochondria. TALE nucleases (TALENs) have been applied to delete chimaeric reading frames and duplicated (redundant) genes but produced complex genomic rearrangements due to the absence of non-homologous end-joining. Here we report the targeted deletion of a conserved mitochondrial gene, nad9, encoding a subunit of respiratory complex I. By generating a large number of TALEN-mediated mitochondrial deletion lines, we isolated, in addition to mutants with rearranged genomes, homochondriomic mutants harbouring clean nad9 deletions. Characterization of the knockout plants revealed impaired complex I biogenesis, male sterility and defects in leaf and flower development. We show that these defects can be restored by expressing a functional Nad9 protein from the nuclear genome, thus creating a synthetic cytoplasmic male sterility system. Our data (1) demonstrate the feasibility of using genome editing to study mitochondrial gene functions by reverse genetics, (2) highlight the role of complex I in plant development and (3) provide proof-of-concept for the construction of synthetic cytoplasmic male sterility systems for hybrid breeding by genome editing.

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.

Targeted introduction of heritable point mutations into the plant mitochondrial genome.

The development of technologies for the genetic manipulation of mitochondrial genomes remains a major challenge. Here we report a method for the targeted introduction of mutations into plant mitochondrial DNA (mtDNA) that we refer to as transcription activator-like effector nuclease (TALEN) gene-drive mutagenesis (GDM), or TALEN-GDM. The method combines TALEN-induced site-specific cleavage of the mtDNA with selection for mutations that confer resistance to the TALEN cut. Applying TALEN-GDM to the tobacco mitochondrial nad9 gene, we isolated a large set of mutants carrying single amino acid substitutions in the Nad9 protein. The mutants could be purified to homochondriomy and stably inherited their edited mtDNA in the expected maternal fashion. TALEN-GDM induces both transitions and transversions, and can access most nucleotide positions within the TALEN binding site. Our work provides an efficient method for targeted mitochondrial genome editing that produces genetically stable, homochondriomic and fertile plants with specific point mutations in their mtDNA.

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.

Improving plant drought tolerance and growth under water limitation through combinatorial engineering of signalling networks.

Agriculture is by far the biggest water consumer on our planet, accounting for 70 per cent of all freshwater withdrawals. Climate change and a growing world population increase pressure on agriculture to use water more efficiently ('more crop per drop'). Water-use efficiency (WUE) and drought tolerance of crops are complex traits that are determined by many physiological processes whose interplay is not well understood. Here, we describe a combinatorial engineering approach to optimize signalling networks involved in the control of stress tolerance. Screening a large population of combinatorially transformed plant lines, we identified a combination of calcium-dependent protein kinase genes that confers enhanced drought stress tolerance and improved growth under water-limiting conditions. Targeted introduction of this gene combination into plants increased plant survival under drought and enhanced growth under water-limited conditions. Our work provides an efficient strategy for engineering complex signalling networks to improve plant performance under adverse environmental conditions, which does not depend on prior understanding of network function.

Control of retrograde signalling by protein import and cytosolic folding stress.

Communication between organelles and the nucleus is essential for fitness and survival. Retrograde signals are cues emitted from the organelles to regulate nuclear gene expression. GENOMES UNCOUPLED1 (GUN1), a protein of unknown function, has emerged as a central integrator, participating in multiple retrograde signalling pathways that collectively regulate the nuclear transcriptome. Here, we show that GUN1 regulates chloroplast protein import through interaction with the import-related chaperone cpHSC70-1. We demonstrated that overaccumulation of unimported precursor proteins (preproteins) in the cytosol causes a GUN phenotype in the wild-type background and enhances the GUN phenotype of the gun1 mutant. Furthermore, we identified the cytosolic HSP90 chaperone complex, induced by overaccumulated preproteins, as a central regulator of photosynthetic gene expression that determines the expression of the GUN phenotype. Taken together, our results suggest a model in which protein import capacity, folding stress and the cytosolic HSP90 complex control retrograde communication.

AtRsgA from Arabidopsis thaliana is important for maturation of the small subunit of the chloroplast ribosome.

Plastid ribosomes are very similar in structure and function to the ribosomes of their bacterial ancestors. Since ribosome biogenesis is not thermodynamically favorable under biological conditions it requires the activity of many assembly factors. Here we have characterized a homolog of bacterial RsgA in Arabidopsis thaliana and show that it can complement the bacterial homolog. Functional characterization of a strong mutant in Arabidopsis revealed that the protein is essential for plant viability, while a weak mutant produced dwarf, chlorotic plants that incorporated immature pre-16S ribosomal RNA into translating ribosomes. Physiological analysis of the mutant plants revealed smaller, but more numerous, chloroplasts in the mesophyll cells, reduction of chlorophyll a and b, depletion of proplastids from the rib meristem and decreased photosynthetic electron transport rate and efficiency. Comparative RNA sequencing and proteomic analysis of the weak mutant and wild-type plants revealed that various biotic stress-related, transcriptional regulation and post-transcriptional modification pathways were repressed in the mutant. Intriguingly, while nuclear- and chloroplast-encoded photosynthesis-related proteins were less abundant in the mutant, the corresponding transcripts were increased, suggesting an elaborate compensatory mechanism, potentially via differentially active retrograde signaling pathways. To conclude, this study reveals a chloroplast ribosome assembly factor and outlines the transcriptomic and proteomic responses of the compensatory mechanism activated during decreased chloroplast function.

Generation and characterization of a collection of knock-down lines for the chloroplast Clp protease complex in tobacco.

Protein degradation in chloroplasts is carried out by a set of proteases that eliminate misfolded, damaged, or superfluous proteins. The ATP-dependent caseinolytic protease (Clp) is the most complex protease in plastids and has been implicated mainly in stromal protein degradation. In contrast, FtsH, a thylakoid membrane-associated metalloprotease, is believed to participate mainly in the degradation of thylakoidal proteins. To determine the role of specific Clp and FtsH subunits in plant growth and development, RNAi lines targeting at least one subunit of each Clp ring and FtsH were generated in tobacco. In addition, mutation of the translation initiation codon was employed to down-regulate expression of the plastid-encoded ClpP1 subunit. These protease lines cover a broad range of reductions at the transcript and protein levels of the targeted genes. A wide spectrum of phenotypes was obtained, including pigment deficiency, alterations in leaf development, leaf variegations, and impaired photosynthesis. When knock-down lines for the different protease subunits were compared, both common and specific phenotypes were observed, suggesting distinct functions of at least some subunits. Our work provides a well-characterized collection of knock-down lines for plastid proteases in tobacco and reveals the importance of the Clp protease in physiology and plant development.

Identification and characterization of a stable intermediate in photosystem I assembly in tobacco.

Photosystem I (PSI) is the most efficient bioenergetic nanomachine in nature and one of the largest membrane protein complexes known. It is composed of 18 protein subunits that bind more than 200 co-factors and prosthetic groups. While the structure and function of PSI have been studied in great detail, very little is known about the PSI assembly process. In this work, we have characterized a PSI assembly intermediate in tobacco plants, which we named PSI*. We found PSI* to contain only a specific subset of the core subunits of PSI. PSI* is particularly abundant in young leaves where active thylakoid biogenesis takes place. Moreover, PSI* was found to overaccumulate in PsaF-deficient mutant plants, and we show that re-initiation of PsaF synthesis promotes the maturation of PSI* into PSI. The attachment of antenna proteins to PSI also requires the transition from PSI* to mature PSI. Our data could provide a biochemical entry point into the challenging investigation of PSI biogenesis and allow us to improve the model for the assembly pathway of PSI in thylakoid membranes of vascular plants.

Photosynthetic complex stoichiometry dynamics in higher plants: environmental acclimation and photosynthetic flux control.

The composition of the photosynthetic apparatus of higher plants is dynamically adjusted to long-term changes in environmental conditions such as growth light intensity and light quality, and to changing metabolic demands for ATP and NADPH imposed by stresses and leaf aging. By changing photosynthetic complex stoichiometry, a long-term imbalance between the photosynthetic production of ATP and NADPH and their metabolic consumption is avoided, and cytotoxic side reactions are minimized. Otherwise, an excess capacity of the light reactions, relative to the demands of primary metabolism, could result in a disturbance of cellular redox homeostasis and an increased production of reactive oxygen species, leading to the destruction of the photosynthetic apparatus and the initiation of cell death programs. In this review, changes of the abundances of the different constituents of the photosynthetic apparatus in response to environmental conditions and during leaf ontogenesis are summarized. The contributions of the different photosynthetic complexes to photosynthetic flux control and the regulation of electron transport are discussed.

Reverse genetics in complex multigene operons by co-transformation of the plastid genome and its application to the open reading frame previously designated psbN.

Reverse genetics approaches have contributed enormously to the elucidation of gene functions in plastid genomes and the determination of structure-function relationships in chloroplast multiprotein complexes. Gene knock-outs are usually performed by disrupting the reading frame of interest with a selectable marker cassette. Site-directed mutagenesis is done by placing the marker into the adjacent intergenic spacer and relying on co-integration of the desired mutation by homologous recombination. These strategies are not applicable to genes residing in large multigene operons or other gene-dense genomic regions, because insertion of the marker cassette into an operon-internal gene or into the nearest intergenic spacer is likely to interfere with expression of adjacent genes in the operon or disrupt cis-elements for the expression of neighboring genes and operons. Here we have explored the possibility of using a co-transformation strategy to mutate a small gene of unknown function (psbN) that is embedded in a complex multigene operon. Although inactivation of psbN resulted in strong impairment of photosynthesis, homoplasmic knock-out lines were readily recovered by co-transformation with a selectable marker integrating >38 kb away from the targeted psbN. Our results suggest co-transformation as a suitable strategy for the functional analysis of plastid genes and operons, which allows the recovery of unselected homoplasmic mutants even if the introduced mutations entail a significant selective disadvantage. Moreover, our data provide evidence for involvement of the psbN gene product in the biogenesis of both photosystem I and photosystem II. We therefore propose to rename the gene product 'photosystem biogenesis factor 1' and the gene pbf1.

The contributions of wobbling and superwobbling to the reading of the genetic code.

Reduced bacterial genomes and most genomes of cell organelles (chloroplasts and mitochondria) do not encode the full set of 32 tRNA species required to read all triplets of the genetic code according to the conventional wobble rules. Superwobbling, in which a single tRNA species that contains a uridine in the wobble position of the anticodon reads an entire four-fold degenerate codon box, has been suggested as a possible mechanism for how tRNA sets can be reduced. However, the general feasibility of superwobbling and its efficiency in the various codon boxes have remained unknown. Here we report a complete experimental assessment of the decoding rules in a typical prokaryotic genetic system, the plastid genome. By constructing a large set of transplastomic knock-out mutants for pairs of isoaccepting tRNA species, we show that superwobbling occurs in all codon boxes where it is theoretically possible. Phenotypic characterization of the transplastomic mutant plants revealed that the efficiency of superwobbling varies in a codon box-dependent manner, but--contrary to previous suggestions--it is independent of the number of hydrogen bonds engaged in codon-anticodon interaction. Finally, our data provide experimental evidence of the minimum tRNA set comprising 25 tRNA species, a number lower than previously suggested. Our results demonstrate that all triplets with pyrimidines in third codon position are dually decoded: by a tRNA species utilizing standard base pairing or wobbling and by a second tRNA species employing superwobbling. This has important implications for the interpretation of the genetic code and will aid the construction of synthetic genomes with a minimum-size translational apparatus.

The plastid genome-encoded Ycf4 protein functions as a nonessential assembly factor for photosystem I in higher plants.

Photosystem biogenesis in the thylakoid membrane is a highly complicated process that requires the coordinated assembly of nucleus-encoded and chloroplast-encoded protein subunits as well as the insertion of hundreds of cofactors, such as chromophores (chlorophylls, carotenoids) and iron-sulfur clusters. The molecular details of the assembly process and the identity and functions of the auxiliary factors involved in it are only poorly understood. In this work, we have characterized the chloroplast genome-encoded ycf4 (for hypothetical chloroplast reading frame no. 4) gene, previously shown to encode a protein involved in photosystem I (PSI) biogenesis in the unicellular green alga Chlamydomonas reinhardtii. Using stable transformation of the chloroplast genome, we have generated ycf4 knockout plants in the higher plant tobacco (Nicotiana tabacum). Although these mutants are severely affected in their photosynthetic performance, they are capable of photoautotrophic growth, demonstrating that, different from Chlamydomonas, the ycf4 gene product is not essential for photosynthesis. We further show that ycf4 knockout plants are specifically deficient in PSI accumulation. Unaltered expression of plastid-encoded PSI genes and biochemical analyses suggest a posttranslational action of the Ycf4 protein in the PSI assembly process. With increasing leaf age, the contents of Ycf4 and Y3IP1, another auxiliary factor involved in PSI assembly, decrease strongly, whereas PSI contents remain constant, suggesting that PSI is highly stable and that its biogenesis is restricted to young leaves.

The plastid-specific ribosomal proteins of Arabidopsis thaliana can be divided into non-essential proteins and genuine ribosomal proteins.

Plastid translation occurs on bacterial-type 70S ribosomes consisting of a large (50S) subunit and a small (30S) subunit. The vast majority of plastid ribosomal proteins have orthologs in bacteria. In addition, plastids also possess a small set of unique ribosomal proteins, so-called plastid-specific ribosomal proteins (PSRPs). The functions of these PSRPs are unknown, but, based on structural studies, it has been proposed that they may represent accessory proteins involved in translational regulation. Here we have investigated the functions of five PSRPs using reverse genetics in the model plant Arabidopsis thaliana. By analyzing T-DNA insertion mutants and RNAi lines, we show that three PSRPs display characteristics of genuine ribosomal proteins, in that down-regulation of their expression led to decreased accumulation of the 30S or 50S subunit of the plastid ribosomes, resulting in plastid translational deficiency. In contrast, two other PSRPs can be knocked out without visible or measurable phenotypic consequences. Our data suggest that PSRPs fall into two types: (i) PSRPs that have a structural role in the ribosome and are bona fide ribosomal proteins, and (ii) non-essential PSRPs that are not required for stable ribosome accumulation and translation under standard greenhouse conditions.

Nonessential plastid-encoded ribosomal proteins in tobacco: a developmental role for plastid translation and implications for reductive genome evolution.

Plastid genomes of higher plants contain a conserved set of ribosomal protein genes. Although plastid translational activity is essential for cell survival in tobacco (Nicotiana tabacum), individual plastid ribosomal proteins can be nonessential. Candidates for nonessential plastid ribosomal proteins are ribosomal proteins identified as nonessential in bacteria and those whose genes were lost from the highly reduced plastid genomes of nonphotosynthetic plastid-bearing lineages (parasitic plants, apicomplexan protozoa). Here we report the reverse genetic analysis of seven plastid-encoded ribosomal proteins that meet these criteria. We have introduced knockout alleles for the corresponding genes into the tobacco plastid genome. Five of the targeted genes (ribosomal protein of the large subunit22 [rpl22], rpl23, rpl32, ribosomal protein of the small subunit3 [rps3], and rps16) were shown to be essential even under heterotrophic conditions, despite their loss in at least some parasitic plastid-bearing lineages. This suggests that nonphotosynthetic plastids show elevated rates of gene transfer to the nuclear genome. Knockout of two ribosomal protein genes, rps15 and rpl36, yielded homoplasmic transplastomic mutants, thus indicating nonessentiality. Whereas Δrps15 plants showed only a mild phenotype, Δrpl36 plants were severely impaired in photosynthesis and growth and, moreover, displayed greatly altered leaf morphology. This finding provides strong genetic evidence that chloroplast translational activity influences leaf development, presumably via a retrograde signaling pathway.

Elimination of a group II intron from a plastid gene causes a mutant phenotype.

Group II introns are found in bacteria and cell organelles (plastids, mitochondria) and are thought to represent the evolutionary ancestors of spliceosomal introns. It is generally believed that group II introns are selfish genetic elements that do not have any function. Here, we have scrutinized this assumption by analyzing two group II introns that interrupt a plastid gene (ycf3) involved in photosystem assembly. Using stable transformation of the plastid genome, we have generated mutant plants that lack either intron 1 or intron 2 or both. Interestingly, the deletion of intron 1 caused a strong mutant phenotype. We show that the mutants are deficient in photosystem I and that this deficiency is directly related to impaired ycf3 function. We further show that, upon deletion of intron 1, the splicing of intron 2 is strongly inhibited. Our data demonstrate that (i) the loss of a group II intron is not necessarily phenotypically neutral and (ii) the splicing of one intron can depend on the presence of another.