Riboswitch-mediated inducible expression of an astaxanthin biosynthetic operon in plastids.

The high-value carotenoid astaxanthin (3,3'-dihydroxy-ß,ß-carotene-4,4'-dione) is one of the most potent antioxidants in nature. In addition to its large-scale use in fish farming, the pigment has applications as a food supplement and an active ingredient in cosmetics and in pharmaceuticals for the treatment of diseases linked to reactive oxygen species. The biochemical pathway for astaxanthin synthesis has been introduced into seed plants, which do not naturally synthesize this pigment, by nuclear and plastid engineering. The highest accumulation rates have been achieved in transplastomic plants, but massive production of astaxanthin has resulted in severe growth retardation. What limits astaxanthin accumulation levels and what causes the mutant phenotype is unknown. Here, we addressed these questions by making astaxanthin synthesis in tobacco (Nicotiana tabacum) plastids inducible by a synthetic riboswitch. We show that, already in the uninduced state, astaxanthin accumulates to similarly high levels as in transplastomic plants expressing the pathway constitutively. Importantly, the inducible plants displayed wild-type-like growth properties and riboswitch induction resulted in a further increase in astaxanthin accumulation. Our data suggest that the mutant phenotype associated with constitutive astaxanthin synthesis is due to massive metabolite turnover, and indicate that astaxanthin accumulation is limited by the sequestration capacity of the plastid.

A Sec14 domain protein is required for photoautotrophic growth and chloroplast vesicle formation in Arabidopsis thaliana.

In eukaryotic photosynthetic organisms, the conversion of solar into chemical energy occurs in thylakoid membranes in the chloroplast. How thylakoid membranes are formed and maintained is poorly understood. However, previous observations of vesicles adjacent to the stromal side of the inner envelope membrane of the chloroplast suggest a possible role of membrane transport via vesicle trafficking from the inner envelope to the thylakoids. Here we show that the model plant Arabidopsis thaliana has a chloroplast-localized Sec14-like protein (CPSFL1) that is necessary for photoautotrophic growth and vesicle formation at the inner envelope membrane of the chloroplast. The cpsfl1 mutants are seedling lethal, show a defect in thylakoid structure, and lack chloroplast vesicles. Sec14 domain proteins are found only in eukaryotes and have been well characterized in yeast, where they regulate vesicle budding at the trans-Golgi network. Like the yeast Sec14p, CPSFL1 binds phosphatidylinositol phosphates (PIPs) and phosphatidic acid (PA) and acts as a phosphatidylinositol transfer protein in vitro, and expression of Arabidopsis CPSFL1 can complement the yeast sec14 mutation. CPSFL1 can transfer PIP into PA-rich membrane bilayers in vitro, suggesting that CPSFL1 potentially facilitates vesicle formation by trafficking PA and/or PIP, known regulators of membrane trafficking between organellar subcompartments. These results underscore the role of vesicles in thylakoid biogenesis and/or maintenance. CPSFL1 appears to be an example of a eukaryotic cytosolic protein that has been coopted for a function in the chloroplast, an organelle derived from endosymbiosis of a cyanobacterium.

PGRL1 is the elusive ferredoxin-plastoquinone reductase in photosynthetic cyclic electron flow.

During plant photosynthesis, photosystems I (PSI) and II (PSII), located in the thylakoid membranes of the chloroplast, use light energy to mobilize electron transport. Different modes of electron flow exist. Linear electron flow is driven by both photosystems and generates ATP and NADPH, whereas cyclic electron flow (CEF) is driven by PSI alone and generates ATP only. Two variants of CEF exist in flowering plants, of which one is sensitive to antimycin A (AA) and involves the two thylakoid proteins, PGR5 and PGRL1. However, neither the mechanism nor the site of reinjection of electrons from ferredoxin into the thylakoid electron transport chain during AA-sensitive CEF is known. Here, we show that PGRL1 accepts electrons from ferredoxin in a PGR5-dependent manner and reduces quinones in an AA-sensitive fashion. PGRL1 activity itself requires several redox-active cysteine residues and a Fe-containing cofactor. We therefore propose that PGRL1 is the elusive ferredoxin-plastoquinone reductase (FQR).

Highly Resolved Systems Biology to Dissect the Etioplast-to-Chloroplast Transition in Tobacco Leaves.

Upon exposure to light, plant cells quickly acquire photosynthetic competence by converting pale etioplasts into green chloroplasts. This developmental transition involves the de novo biogenesis of the thylakoid system and requires reprogramming of metabolism and gene expression. Etioplast-to-chloroplast differentiation involves massive changes in plastid ultrastructure, but how these changes are connected to specific changes in physiology, metabolism, and expression of the plastid and nuclear genomes is poorly understood. Here, we describe a new experimental system in the dicotyledonous model plant tobacco (Nicotiana tabacum) that allows us to study the leaf deetiolation process at the systems level. We have determined the accumulation kinetics of photosynthetic complexes, pigments, lipids, and soluble metabolites and recorded the dynamic changes in plastid ultrastructure and in the nuclear and plastid transcriptomes. Our data describe the greening process at high temporal resolution, resolve distinct genetic and metabolic phases during deetiolation, and reveal numerous candidate genes that may be involved in light-induced chloroplast development and thylakoid biogenesis.

The photosynthesis affected mutant68-like protein evolved from a PSII assembly factor to mediate assembly of the chloroplast NAD(P)H dehydrogenase complex in Arabidopsis.

In vascular plants, the chloroplast NAD(P)H dehydrogenase complex (NDH-C) is assembled from five distinct subcomplexes, the membrane-spanning (subM) and the luminal (subL) subcomplexes, as well as subA, subB, and subE. The assembly process itself is poorly understood. Vascular plant genomes code for two related intrinsic thylakoid proteins, photosynthesis-affected mutant68 (PAM68), a photosystem II assembly factor, and photosynthesis-affected mutant68-like (PAM68L). As we show here, inactivation of Arabidopsis thaliana PAM68L in the pam68l-1 mutant identifies PAM68L as an NDH-C assembly factor. The mutant lacks functional NDH holocomplexes and accumulates three distinct NDH-C assembly intermediates (subB, subM, and subA+L), which are also found in mutants defective in subB assembly (ndf5) or subM expression (chlororespiratory reduction4-3 mutant). NDH-C assembly in the cyanobacterium Synechocystis sp PCC 6803 and the moss Physcomitrella patens does not require PAM68 proteins, as demonstrated by the analysis of knockout lines for the single-copy PAM68 genes in these species. We conclude that PAM68L mediates the attachment of subB- and subM-containing intermediates to a complex that contains subA and subL. The evolutionary appearance of subL and PAM68L during the transition from mosses like P. patens to flowering plants suggests that the associated increase in the complexity of the NDH-C might have been facilitated by the recruitment of evolutionarily novel assembly factors like PAM68L.

Arabidopsis CURVATURE THYLAKOID1 proteins modify thylakoid architecture by inducing membrane curvature.

Chloroplasts of land plants characteristically contain grana, cylindrical stacks of thylakoid membranes. A granum consists of a core of appressed membranes, two stroma-exposed end membranes, and margins, which connect pairs of grana membranes at their lumenal sides. Multiple forces contribute to grana stacking, but it is not known how the extreme curvature at margins is generated and maintained. We report the identification of the CURVATURE THYLAKOID1 (CURT1) protein family, conserved in plants and cyanobacteria. The four Arabidopsis thaliana CURT1 proteins (CURT1A, B, C, and D) oligomerize and are highly enriched at grana margins. Grana architecture is correlated with the CURT1 protein level, ranging from flat lobe-like thylakoids with considerably fewer grana margins in plants without CURT1 proteins to an increased number of membrane layers (and margins) in grana at the expense of grana diameter in overexpressors of CURT1A. The endogenous CURT1 protein in the cyanobacterium Synechocystis sp PCC6803 can be partially replaced by its Arabidopsis counterpart, indicating that the function of CURT1 proteins is evolutionary conserved. In vitro, Arabidopsis CURT1A proteins oligomerize and induce tubulation of liposomes, implying that CURT1 proteins suffice to induce membrane curvature. We therefore propose that CURT1 proteins modify thylakoid architecture by inducing membrane curvature at grana margins.

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.

The thylakoid proton antiporter KEA3 regulates photosynthesis in response to the chloroplast energy status.

Plant photosynthesis contains two functional modules, the light-driven reactions in the thylakoid membrane and the carbon-fixing reactions in the chloroplast stroma. In nature, light availability for photosynthesis often undergoes massive and rapid fluctuations. Efficient and productive use of such variable light supply requires an instant crosstalk and rapid synchronization of both functional modules. Here, we show that this communication involves the stromal exposed C-terminus of the thylakoid K+-exchange antiporter KEA3, which regulates the ΔpH across the thylakoid membrane and therefore pH-dependent photoprotection. By combining in silico, in vitro, and in vivo approaches, we demonstrate that the KEA3 C-terminus senses the energy state of the chloroplast in a pH-dependent manner and regulates transport activity in response. Together our data pinpoint a regulatory feedback loop by which the stromal energy state orchestrates light capture and photoprotection via multi-level regulation of KEA3.

Role of plastid protein phosphatase TAP38 in LHCII dephosphorylation and thylakoid electron flow.

Short-term changes in illumination elicit alterations in thylakoid protein phosphorylation and reorganization of the photosynthetic machinery. Phosphorylation of LHCII, the light-harvesting complex of photosystem II, facilitates its relocation to photosystem I and permits excitation energy redistribution between the photosystems (state transitions). The protein kinase STN7 is required for LHCII phosphorylation and state transitions in the flowering plant Arabidopsis thaliana. LHCII phosphorylation is reversible, but extensive efforts to identify the protein phosphatase(s) that dephosphorylate LHCII have been unsuccessful. Here, we show that the thylakoid-associated phosphatase TAP38 is required for LHCII dephosphorylation and for the transition from state 2 to state 1 in A. thaliana. In tap38 mutants, thylakoid electron flow is enhanced, resulting in more rapid growth under constant low-light regimes. TAP38 gene overexpression markedly decreases LHCII phosphorylation and inhibits state 1-->2 transition, thus mimicking the stn7 phenotype. Furthermore, the recombinant TAP38 protein is able, in an in vitro assay, to directly dephosphorylate LHCII. The dependence of LHCII dephosphorylation upon TAP38 dosage, together with the in vitro TAP38-mediated dephosphorylation of LHCII, suggests that TAP38 directly acts on LHCII. Although reversible phosphorylation of LHCII and state transitions are crucial for plant fitness under natural light conditions, LHCII hyperphosphorylation associated with an arrest of photosynthesis in state 2 due to inactivation of TAP38 improves photosynthetic performance and plant growth under state 2-favoring light conditions.

De-etiolation-induced protein 1 (DEIP1) mediates assembly of the cytochrome b6f complex in Arabidopsis.

The conversion of light energy to chemical energy by photosynthesis requires the concerted action of large protein complexes in the thylakoid membrane. Recent work has provided fundamental insights into the three-dimensional structure of these complexes, but how they are assembled from hundreds of parts remains poorly understood. Particularly little is known about the biogenesis of the cytochrome b6f complex (Cytb6f), the redox-coupling complex that interconnects the two photosystems. Here we report the identification of a factor that guides the assembly of Cytb6f in thylakoids of chloroplasts. The protein, DE-ETIOLATION-INDUCED PROTEIN 1 (DEIP1), resides in the thylakoid membrane and is essential for photoautotrophic growth. Knock-out mutants show a specific loss of Cytb6f, and are defective in complex assembly. We demonstrate that DEIP1 interacts with the two cytochrome subunits of the complex, PetA and PetB, and mediates the assembly of intermediates in Cytb6f biogenesis. The identification of DEIP1 provides an entry point into the study of the assembly pathway of a crucial complex in photosynthetic electron transfer.

Horizontal genome transfer by cell-to-cell travel of whole organelles.

Recent work has revealed that both plants and animals transfer genomes between cells. In plants, horizontal transfer of entire plastid, mitochondrial, or nuclear genomes between species generates new combinations of nuclear and organellar genomes, or produces novel species that are allopolyploid. The mechanisms of genome transfer between cells are unknown. Here, we used grafting to identify the mechanisms involved in plastid genome transfer from plant to plant. We show that during proliferation of wound-induced callus, plastids dedifferentiate into small, highly motile, amoeboid organelles. Simultaneously, new intercellular connections emerge by localized cell wall disintegration, forming connective pores through which amoeboid plastids move into neighboring cells. Our work uncovers a pathway of organelle movement from cell to cell and provides a mechanistic framework for horizontal genome transfer.

Cytochrome b6f is a dimeric protochlorophyll a binding complex in etioplasts.

The cytochrome b6f complex is a dimeric protein complex that is of central importance for photosynthesis to carry out light driven electron and proton transfer in chloroplasts. One molecule of chlorophyll a was found to associate per cytochrome b6f monomer and the structural or functional importance of this is discussed. We show that etioplasts which are devoid of chlorophyll a already contain dimeric cytochrome b6f. However, the phytylated chlorophyll precursor protochlorophyll a, and not chlorophyll a, is associated with subunit b6. The data imply that a phytylated tetrapyrrol is an essential structural requirement for assembly of cytochrome b6f.

Absence of Complex I Is Associated with Diminished Respiratory Chain Function in European Mistletoe.

Parasitism is a life history strategy found across all domains of life whereby nutrition is obtained from a host. It is often associated with reductive evolution of the genome, including loss of genes from the organellar genomes [1, 2]. In some unicellular parasites, the mitochondrial genome (mitogenome) has been lost entirely, with far-reaching consequences for the physiology of the organism [3, 4]. Recently, mitogenome sequences of several species of the hemiparasitic plant mistletoe (Viscum sp.) have been reported [5, 6], revealing a striking loss of genes not seen in any other multicellular eukaryotes. In particular, the nad genes encoding subunits of respiratory complex I are all absent and other protein-coding genes are also lost or highly diverged in sequence, raising the question what remains of the respiratory complexes and mitochondrial functions. Here we show that oxidative phosphorylation (OXPHOS) in European mistletoe, Viscum album, is highly diminished. Complex I activity and protein subunits of complex I could not be detected. The levels of complex IV and ATP synthase were at least 5-fold lower than in the non-parasitic model plant Arabidopsis thaliana, whereas alternative dehydrogenases and oxidases were higher in abundance. Carbon flux analysis indicates that cytosolic reactions including glycolysis are greater contributors to ATP synthesis than the mitochondrial tricarboxylic acid (TCA) cycle. Our results describe the extreme adjustments in mitochondrial functions of the first reported multicellular eukaryote without complex I.

Update on chloroplast research: new tools, new topics, and new trends.

Chloroplasts, the green differentiation form of plastids, are the sites of photosynthesis and other important plant functions. Genetic and genomic technologies have greatly boosted the rate of discovery and functional characterization of chloroplast proteins during the past decade. Indeed, data obtained using high-throughput methodologies, in particular proteomics and transcriptomics, are now routinely used to assign functions to chloroplast proteins. Our knowledge of many chloroplast processes, notably photosynthesis and photorespiration, has reached such an advanced state that biotechnological approaches to crop improvement now seem feasible. Meanwhile, efforts to identify the entire complement of chloroplast proteins and their interactions are progressing rapidly, making the organelle a prime target for systems biology research in plants.

Optimizing photosynthesis under fluctuating light: the role of the Arabidopsis STN7 kinase.

Optimal photosynthetic performance requires that equal amounts of light are absorbed by photosystem II (PSII) and photosystem I (PSi), which are functionally linked through the photosynthetic electron transport chain. However, photosynthetic organisms must cope with light conditions that lead to the preferential stimulation of one or the other of the photosystems. Plants react to such imbalances by mounting acclimation responses that redistribute excitation energy between photosystems and restore the photosynthetic redox poise. in the short term, this involves the so-called state transition process, which, over periods of minutes, alters the antennal cross-sections of the photosystems through the reversible association of a mobile fraction of light-harvesting complex II (LHCII) with PSI or PSII. Longer-lasting changes in light quality initiate a long-term response (LTr), occurring on a timescale of hours to days, that redresses imbalances in excitation energy by changing the relative amounts of the two photosystems. Despite the differences in their timescales of action, state transitions and LTr are both triggered by the redox state of the plastoquinone (PQ) pool, via the activation of the thylakoid kinase STN7, which appears to act as a common redox sensor and/or signal transducer for both responses. This review highlights recent findings concerning the role of STN7 in coordinating short- and long-term photosynthetic acclimation responses.

Chloroplast proteins without cleavable transit peptides: rare exceptions or a major constituent of the chloroplast proteome?

Most chloroplast proteins (cp proteins) are nucleus-encoded, synthesized on cytosolic ribosomes as precursor proteins containing a presequence (cTP), and post-translationally imported via the Tic/Toc complex into the organelle, where the cTP is removed. Only a few unambiguous instances of cp proteins that do not require cTPs (non-canonical cp proteins) have been reported so far. However, the survey of data from large-scale proteomic studies presented here suggests that the fraction of such proteins in the total cp proteome might be as large as approximately 30%. To explore this discrepancy, we chose a representative set of 28 putative non-canonical cp proteins, and used in vitro import and Red Fluorescent Protein (RFP)-fusion assays to determine their sub-cellular destinations. Four proteins, including embryo defective 1211, glycolate oxidase 2, protein disulfide isomerase-like protein (PDII), and a putative glutathione S-transferase, could be unambiguously assigned to the chloroplast. Several others ('potential cp proteins') were found to be imported into chloroplasts in vitro, but failed to localize to the organelle when RFP was fused to their C-terminal ends. Extrapolations suggest that the fraction of cp proteins that enter the inner compartments of the organelle, although they lack a cTP, might be as large as 11.4% of the total cp proteome. Our data also support the idea that cytosolic proteins that associate with the cp outer membrane might account for false positive cp proteins obtained in earlier studies.

Arabidopsis STN7 kinase provides a link between short- and long-term photosynthetic acclimation.

Flowering plants control energy allocation to their photosystems in response to light quality changes. This includes the phosphorylation and migration of light-harvesting complex II (LHCII) proteins (state transitions or short-term response) as well as long-term alterations in thylakoid composition (long-term response or LTR). Both responses require the thylakoid protein kinase STN7. Here, we show that the signaling pathways triggering state transitions and LTR diverge at, or immediately downstream from, STN7. Both responses require STN7 activity that can be regulated according to the plastoquinone pool redox state. However, LTR signaling does not involve LHCII phosphorylation or any other state transition step. State transitions appear to play a prominent role in flowering plants, and the ability to perform state transitions becomes critical for photosynthesis in Arabidopsis thaliana mutants that are impaired in thylakoid electron transport but retain a functional LTR. Our data imply that STN7-dependent phosphorylation of an as yet unknown thylakoid protein triggers LTR signaling events, whereby an involvement of the TSP9 protein in the signaling pathway could be excluded. The LTR signaling events then ultimately regulate in chloroplasts the expression of photosynthesis-related genes on the transcript level, whereas expression of nuclear-encoded proteins is regulated at multiple levels, as indicated by transcript and protein profiling in LTR mutants.