Chilling stress drives organ-specific transcriptional cascades and dampens diurnal oscillation in tomato.

Improving chilling tolerance in cold-sensitive crops, e.g. tomato, requires knowledge of the early molecular response to low temperature in these under-studied species. To elucidate early responding processes and regulators, we captured the transcriptional response at 30 minutes and 3 hours in the shoots and at 3 hours in the roots of tomato post-chilling from 24°C to 4°C. We used a pre-treatment control and a concurrent ambient temperature control to reveal that majority of the differential expression between cold and ambient conditions is due to severely compressed oscillation of a large set of diurnally regulated genes in both the shoots and roots. This compression happens within 30 minutes of chilling, lasts for the duration of cold treatment, and is relieved within 3 hours of return to ambient temperatures. Our study also shows that the canonical ICE1/CAMTA-to-CBF cold response pathway is active in the shoots, but not in the roots. Chilling stress induces synthesis of known cryoprotectants (trehalose and polyamines), in a CBF-independent manner, and induction of multiple genes encoding proteins of photosystems I and II. This study provides nuanced insights into the organ-specific response in a chilling sensitive plant, as well as the genes influenced by an interaction of chilling response and the circadian clock.

Quantification of energy-converting protein complexes in plant thylakoid membranes.

Knowledge about the exact abundance and ratio of photosynthetic protein complexes in thylakoid membranes is central to understanding structure-function relationships in energy conversion. Recent modeling approaches for studying light harvesting and electron transport reactions rely on quantitative information on the constituent complexes in thylakoid membranes. Over the last decades several quantitative methods have been established and refined, enabling precise stoichiometric information on the five main energy-converting building blocks in the thylakoid membrane: Light-harvesting complex II (LHCII), Photosystem II (PSII), Photosystem I (PSI), cytochrome b6f complex (cyt b6f complex), and ATPase. This paper summarizes a few quantitative spectroscopic and biochemical methods that are currently available for quantification of plant thylakoid protein complexes. Two new methods are presented for quantification of LHCII and the cyt b6f complex, which agree well with established methods. In addition, recent improvements in mass spectrometry (MS) allow deeper compositional information on thylakoid membranes. The comparison between mass spectrometric and more classical protein quantification methods shows similar quantities of complexes, confirming the potential of thylakoid protein complex quantification by MS. The quantitative information on PSII, PSI, and LHCII reveal that about one third of LHCII must be associated with PSI for a balanced light energy absorption by the two photosystems.

Photosystem stoichiometry adjustment is a photoreceptor-mediated process in Arabidopsis.

Plant growth under spectrally-enriched low light conditions leads to adjustment in the relative abundance of the two photosystems in an acclimatory response known as photosystem stoichiometry adjustment. Adjustment of photosystem stoichiometry improves the quantum efficiency of photosynthesis but how this process perceives light quality changes and how photosystem amount is regulated remain largely unknown. By using a label-free quantitative mass spectrometry approach in Arabidopsis here we show that photosystem stoichiometry adjustment is primarily driven by the regulation of photosystem I content and that this forms the major thylakoid proteomic response under light quality. Using light and redox signaling mutants, we further show that the light quality-responsive accumulation of photosystem I gene transcripts and proteins requires phytochrome B photoreceptor but not plastoquinone redox signaling as previously suggested. In far-red light, the increased acceptor side limitation might deplete active photosystem I pool, further contributing to the adjustment of photosystem stoichiometry.

Thiol redox switches regulate the oligomeric state of cyanobacterial Rre1, RpaA and RpaB response regulators.

Cyanobacteria employ two-component sensor-response regulator systems to monitor and respond to environmental challenges. The response regulators RpaA, RpaB, Rre1 and RppA are integral to circadian clock function and abiotic stress acclimation in cyanobacteria. RpaA, RpaB and Rre1 are known to interact with ferredoxin or thioredoxin, raising the possibility of their thiol regulation. Here, we report that Synechocystis sp. PCC 6803 Rre1, RpaA and RpaB exist as higher-order oligomers under oxidising conditions and that reduced thioredoxin A converts them to monomers. We further show that these response regulators contain redox-responsive cysteine residues with an Em7 around -300 mV. These findings suggest a direct thiol modulation of the activity of these response regulators, independent of their cognate sensor kinases.

Transcription initiation as a control point in plastid gene expression.

The extensive processing and protein-assisted stabilization of transcripts have been taken as evidence for a viewpoint that the control of gene expression had shifted entirely in evolution from transcriptional in the bacterial endosymbiont to posttranscriptional in the plastid. This suggestion is however at odds with many observations on plastid gene transcription. Chloroplasts of flowering plants and mosses contain two or more RNA polymerases with distinct promoter preference and division of labor for the coordinated synthesis of plastid RNAs. Plant and algal plastids further possess multiple nonredundant sigma factors that function as transcription initiation factors. The controlled accumulation of plastid sigma factors and modification of their activity by sigma-binding proteins and phosphorylation constitute additional transcriptional regulatory strategies. Plant and algal plastids also contain dedicated one- or two-component transcriptional regulators. Transcription initiation thus continues to form a critical control point at which varied developmental and environmental signals intersect with plastid gene expression.

Regulation of Phaeodactylum plastid gene transcription by redox, light, and circadian signals.

Diatoms are a diverse group of photosynthetic unicellular algae with a plastid of red-algal origin. As prolific primary producers in the ocean, diatoms fix as much carbon as all rainforests combined. The molecular mechanisms that contribute to the high photosynthetic productivity and ecological success of diatoms are however not yet fully understood. Using the model diatom Phaeodactylum tricornutum, here we show rhythmic transcript accumulation of plastid psaA, psbA, petB, and atpB genes as driven by a free running circadian clock. Treatment with the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea overrides the circadian signal by markedly downregulating transcription of psaA, petB, and atpB genes but not the psbA gene. Changes in light quantity produce little change in plastid gene transcription while the effect of light quality seems modest with only the psaA gene responding in a pattern that is dependent on the redox state of the plastoquinone pool. The significance of these plastid transcriptional responses and the identity of the underlying genetic control systems are discussed with relevance to diatom photosynthetic acclimation.

Plastocyanin is the long-range electron carrier between photosystem II and photosystem I in plants.

In photosynthetic electron transport, large multiprotein complexes are connected by small diffusible electron carriers, the mobility of which is challenged by macromolecular crowding. For thylakoid membranes of higher plants, a long-standing question has been which of the two mobile electron carriers, plastoquinone or plastocyanin, mediates electron transport from stacked grana thylakoids where photosystem II (PSII) is localized to distant unstacked regions of the thylakoids that harbor PSI. Here, we confirm that plastocyanin is the long-range electron carrier by employing mutants with different grana diameters. Furthermore, our results explain why higher plants have a narrow range of grana diameters since a larger diffusion distance for plastocyanin would jeopardize the efficiency of electron transport. In the light of recent findings that the lumen of thylakoids, which forms the diffusion space of plastocyanin, undergoes dynamic swelling/shrinkage, this study demonstrates that plastocyanin diffusion is a crucial regulatory element of plant photosynthetic electron transport.

An evolutionarily conserved iron-sulfur cluster underlies redox sensory function of the Chloroplast Sensor Kinase.

Photosynthetic efficiency depends on equal light energy conversion by two spectrally distinct, serially-connected photosystems. The redox state of the plastoquinone pool, located between the two photosystems, is a key regulatory signal that initiates acclimatory changes in the relative abundance of photosystems. The Chloroplast Sensor Kinase (CSK) links the plastoquinone redox signal with photosystem gene expression but the mechanism by which it monitors the plastoquinone redox state is unclear. Here we show that the purified Arabidopsis and Phaeodactylum CSK and the cyanobacterial CSK homologue, Histidine kinase 2 (Hik2), are iron-sulfur proteins. The Fe-S cluster of CSK is further revealed to be a high potential redox-responsive [3Fe-4S] center. CSK responds to redox agents with reduced plastoquinone suppressing its autokinase activity. Redox changes within the CSK iron-sulfur cluster translate into conformational changes in the protein fold. These results provide key insights into redox signal perception and propagation by the CSK-based chloroplast two-component system.

Stoichiometry of protein complexes in plant photosynthetic membranes.

Hetero-oligomeric membrane protein complexes form the electron transport chain (ETC) of oxygenic photosynthesis. The ETC complexes undertake the light-driven vectorial electron and proton transport reactions, which generate energy-rich ATP and electron-rich NADPH molecules for carbon fixation. The rate of photosynthetic electron transport depends on the availability of photons and the relative abundance of electron transport complexes. The relative abundance of the two photosystems, critical for the quantum efficiency of photosynthesis in changing light quality conditions, has been determined successfully by optical methods. Due to the lack of spectroscopic signatures, however, relatively little is known about the stoichiometry of other non-photosystem complexes in plant photosynthetic membrane. Here we determine the ratios of all major thylakoid-bound ETC complexes in Arabidopsis by a label-free quantitative mass spectrometry technique. The calculated stoichiometries are consistent with known subunit composition of complexes and current estimates of photosystem and cytochrome b6f concentrations. The implications of these stoichiometries for photosynthetic light harvesting and the partitioning of electrons between the linear and cyclic electron transport pathways of photosynthesis are discussed.

Sigma factor 1 in chloroplast gene transcription and photosynthetic light acclimation.

Sigma factors are dissociable subunits of bacterial RNA polymerase that ensure efficient transcription initiation from gene promoters. Owing to their prokaryotic origin, chloroplasts possess a typical bacterial RNA polymerase together with its sigma factor subunit. The higher plant Arabidopsis thaliana contain as many as six sigma factors for the hundred or so of its chloroplast genes. The role of this relatively large number of transcription initiation factors for the miniature chloroplast genome, however, is not fully understood. Using two Arabidopsis T-DNA insertion mutants, we show that sigma factor 1 (SIG1) initiates transcription of a specific subset of chloroplast genes. We further show that the photosynthetic control of PSI reaction center gene transcription requires complementary regulation of the nuclear SIG1 gene at the transcriptional level. This SIG1 gene regulation is dependent on both a plastid redox signal and a light signal transduced by the phytochrome photoreceptor.

Structure-based control of the rate limitation of photosynthetic electron transport.

The 2.5 Å structure of the cytochrome (cyt) b6 f complex provides a basis for control of the rate-limiting electron transfer step of oxygenic photosynthesis associated with the plastoquinol/quinone exchange pathway. Here, a structural change was made at a site containing two proline residues which border the intra-cyt pathway for plastoquinol/quinone exchange. The proline side chains confer a larger aperture for passage of plastoquinol/quinone. Change of these prolines to alanine in the cyanobacterium Synechococcus sp. PCC 7002 results in attenuation of this rate-limiting step, observed by a two-fold reduction in the rate of cell growth, O2 evolution, and plastoquinol-mediated reduction of cyt f. This study demonstrates modification by site-directed mutagenesis of photosynthetic energy transduction based on rational application of information in the atomic structure.

The structural and functional domains of plant thylakoid membranes.

In plants, the stacking of part of the photosynthetic thylakoid membrane generates two main subcompartments: the stacked grana core and unstacked stroma lamellae. However, a third distinct domain, the grana margin, has been postulated but its structural and functional identity remains elusive. Here, an optimized thylakoid fragmentation procedure combined with detailed ultrastructural, biochemical, and functional analyses reveals the distinct composition of grana margins. It is enriched with lipids, cytochrome b6 f complex, and ATPase while depleted in photosystems and light-harvesting complexes. A quantitative method is introduced that is based on Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) and dot immunoblotting for quantifying various photosystem II (PSII) assembly forms in different thylakoid subcompartments. The results indicate that the grana margin functions as a degradation and disassembly zone for photodamaged PSII. In contrast, the stacked grana core region contains fully assembled and functional PSII holocomplexes. The stroma lamellae, finally, contain monomeric PSII as well as a significant fraction of dimeric holocomplexes that identify this membrane area as the PSII repair zone. This structural organization and the heterogeneous PSII distribution support the idea that the stacking of thylakoid membranes leads to a division of labor that establishes distinct membrane areas with specific functions.

Oligomeric states in sodium ion-dependent regulation of cyanobacterial histidine kinase-2.

Two-component signal transduction systems (TCSs) consist of sensor histidine kinases and response regulators. TCSs mediate adaptation to environmental changes in bacteria, plants, fungi and protists. Histidine kinase 2 (Hik2) is a sensor histidine kinase found in all known cyanobacteria and as chloroplast sensor kinase in eukaryotic algae and plants. Sodium ions have been shown to inhibit the autophosphorylation activity of Hik2 that precedes phosphoryl transfer to response regulators, but the mechanism of inhibition has not been determined. We report on the mechanism of Hik2 activation and inactivation probed by chemical cross-linking and size exclusion chromatography together with direct visualisation of the kinase using negative-stain transmission electron microscopy of single particles. We show that the functional form of Hik2 is a higher-order oligomer such as a hexamer or octamer. Increased NaCl concentration converts the active hexamer into an inactive tetramer. The action of NaCl appears to be confined to the Hik2 kinase domain.

Sublocalization of Cytochrome b6f Complexes in Photosynthetic Membranes.

It is well established that the majority of energy-converting photosynthetic protein complexes in plant thylakoid membrane are nonhomogenously distributed between stacked and unstacked membrane regions. Yet, the sublocalization of the central cytochrome b6f complex remains controversial. We present a structural model that explains the variation in cytochrome b6f sublocalization data. Small changes in the distance between adjacent membranes in stacked grana regions either allow or restrict access of cytochrome b6f complexes to grana. If the width of the gap falls below a certain threshold, then the steric hindrance prevents cytochrome b6f access to grana. Evidence is presented that the width of stromal gap is variable, demonstrating that the postulated mechanism can regulate the lateral distribution of the cytochrome b6f complexes.

Surface charge dynamics in photosynthetic membranes and the structural consequences.

The strict stacking of plant photosynthetic membranes into granal structures plays a vital role in energy conversion. The molecular forces that lead to grana stacking, however, are poorly understood. Here we evaluate the interplay between repulsive electrostatic (Fel) and attractive van der Waals (FvdWaals) forces in grana stacking. In contrast to previous reports, we find that the physicochemical balance between attractive and repulsive forces fully explains grana stacking. Extending the force balance analysis to lateral interactions within the oxygen-evolving photosystem II (PSII)-light harvesting complex II (LHCII) supercomplex reveals that supercomplex stability is very sensitive to Fel changes. Fel is highly dynamic, increasing up to 1.7-fold on addition of negative charges by phosphorylation of grana-hosted proteins. We show that this leads to specific destabilization of the supercomplex, and that changes in Fel have contrasting effects on vertical stacking and lateral intramembrane organization. This enables discrete biological control of these central structural features.

A Two-Component Regulatory System in Transcriptional Control of Photosystem Stoichiometry: Redox-Dependent and Sodium Ion-Dependent Phosphoryl Transfer from Cyanobacterial Histidine Kinase Hik2 to Response Regulators Rre1 and RppA.

Two-component systems (TCSs) are ubiquitous signaling units found in prokaryotes. A TCS consists of a sensor histidine kinase and a response regulator protein as signal transducers. These regulatory systems mediate acclimation to various environmental changes by coupling environmental cues to gene expression. Hik2 is a sensor histidine kinase and its gene is found in all cyanobacteria. Hik2 is the homolog of Chloroplast Sensor Kinase (CSK), a protein involved in redox regulation of chloroplast gene expression during changes in light quality in plants and algae. Here we describe biochemical characterization of the signaling mechanism of Hik2 and its phosphotransferase activity. Results presented here indicate that Hik2 undergoes autophosphorylation on a conserved histidine residue, and becomes rapidly dephosphorylated by the action of response regulators Rre1 and RppA. We also show that the autophosphorylation of Hik2 is specifically inhibited by sodium ions.

Probing the nucleotide-binding activity of a redox sensor: two-component regulatory control in chloroplasts.

Two-component signal transduction systems mediate adaptation to environmental changes in bacteria, plants, fungi, and protists. Each two-component system consists of a sensor histidine kinase and a response regulator. Chloroplast sensor kinase (CSK) is a modified sensor histidine kinase found in chloroplasts-photosynthetic organelles of plants and algae. CSK regulates the transcription of chloroplast genes in response to changes in photosynthetic electron transport. In this study, the full-length and truncated forms of Arabidopsis CSK proteins were overexpressed and purified in order to characterise their kinase and redox sensing activities. Our results show that CSK contains a modified kinase catalytic domain that binds ATP with high affinity and forms a quinone adduct that may confer redox sensing activity.

Functional Implications of Photosystem II Crystal Formation in Photosynthetic Membranes.

The structural organization of proteins in biological membranes can affect their function. Photosynthetic thylakoid membranes in chloroplasts have the remarkable ability to change their supramolecular organization between disordered and semicrystalline states. Although the change to the semicrystalline state is known to be triggered by abiotic factors, the functional significance of this protein organization has not yet been understood. Taking advantage of an Arabidopsis thaliana fatty acid desaturase mutant (fad5) that constitutively forms semicrystalline arrays, we systematically test the functional implications of protein crystals in photosynthetic membranes. Here, we show that the change into an ordered state facilitates molecular diffusion of photosynthetic components in crowded thylakoid membranes. The increased mobility of small lipophilic molecules like plastoquinone and xanthophylls has implications for diffusion-dependent electron transport and photoprotective energy-dependent quenching. The mobility of the large photosystem II supercomplexes, however, is impaired, leading to retarded repair of damaged proteins. Our results demonstrate that supramolecular changes into more ordered states have differing impacts on photosynthesis that favor either diffusion-dependent electron transport and photoprotection or protein repair processes, thus fine-tuning the photosynthetic energy conversion.