Poly(ADP-ribose)-binding protein RCD1 is a plant PARylation reader regulated by Photoregulatory Protein Kinases.

Poly(ADP-ribosyl)ation (PARylation) is a reversible post-translational protein modification that has profound regulatory functions in metabolism, development and immunity, and is conserved throughout the eukaryotic lineage. Contrary to metazoa, many components and mechanistic details of PARylation have remained unidentified in plants. Here we present the transcriptional co-regulator RADICAL-INDUCED CELL DEATH1 (RCD1) as a plant PAR-reader. RCD1 is a multidomain protein with intrinsically disordered regions (IDRs) separating its domains. We have reported earlier that RCD1 regulates plant development and stress-tolerance by interacting with numerous transcription factors (TFs) through its C-terminal RST domain. This study suggests that the N-terminal WWE and PARP-like domains, as well as the connecting IDR play an important regulatory role for RCD1 function. We show that RCD1 binds PAR in vitro via its WWE domain and that PAR-binding determines RCD1 localization to nuclear bodies (NBs) in vivo. Additionally, we found that RCD1 function and stability is controlled by Photoregulatory Protein Kinases (PPKs). PPKs localize with RCD1 in NBs and phosphorylate RCD1 at multiple sites affecting its stability. This work proposes a mechanism for negative transcriptional regulation in plants, in which RCD1 localizes to NBs, binds TFs with its RST domain and is degraded after phosphorylation by PPKs.

Pyruvate:ferredoxin oxidoreductase and low abundant ferredoxins support aerobic photomixotrophic growth in cyanobacteria.

The decarboxylation of pyruvate is a central reaction in the carbon metabolism of all organisms. It is catalyzed by the pyruvate:ferredoxin oxidoreductase (PFOR) and the pyruvate dehydrogenase (PDH) complex. Whereas PFOR reduces ferredoxin, the PDH complex utilizes NAD+. Anaerobes rely on PFOR, which was replaced during evolution by the PDH complex found in aerobes. Cyanobacteria possess both enzyme systems. Our data challenge the view that PFOR is exclusively utilized for fermentation. Instead, we show, that the cyanobacterial PFOR is stable in the presence of oxygen in vitro and is required for optimal photomixotrophic growth under aerobic and highly reducing conditions while the PDH complex is inactivated. We found that cells rely on a general shift from utilizing NAD(H)- to ferredoxin-dependent enzymes under these conditions. The utilization of ferredoxins instead of NAD(H) saves a greater share of the Gibbs-free energy, instead of wasting it as heat. This obviously simultaneously decelerates metabolic reactions as they operate closer to their thermodynamic equilibrium. It is common thought that during evolution, ferredoxins were replaced by NAD(P)H due to their higher stability in an oxidizing atmosphere. However, the utilization of NAD(P)H could also have been favored due to a higher competitiveness because of an accelerated metabolism.

The Cytochrome b 6 f Complex Is Not Involved in Cyanobacterial State Transitions.

Photosynthetic organisms must sense and respond to fluctuating environmental conditions in order to perform efficient photosynthesis and to avoid the formation of dangerous reactive oxygen species. The excitation energy arriving at each photosystem permanently changes due to variations in the intensity and spectral properties of the absorbed light. Cyanobacteria, like plants and algae, have developed a mechanism, named "state transitions," that balances photosystem activities. Here, we characterize the role of the cytochrome b 6 f complex and phosphorylation reactions in cyanobacterial state transitions using Synechococcus elongatus PCC 7942 and Synechocystis PCC 6803 as model organisms. First, large photosystem II (PSII) fluorescence quenching was observed in State II, a result that does not appear to be related to energy transfer from PSII to PSI (spillover). This membrane-associated process was inhibited by betaine, Suc, and high concentrations of phosphate. Then, using different chemicals affecting the plastoquinone pool redox state and cytochrome b 6 f activity, we demonstrate that this complex is not involved in state transitions in S. elongatus or Synechocystis PCC6803. Finally, by constructing and characterizing 21 protein kinase and phosphatase mutants and using chemical inhibitors, we demonstrate that phosphorylation reactions are not essential for cyanobacterial state transitions. Thus, signal transduction is completely different in cyanobacterial and plant (green alga) state transitions.

Proteomics of cyanobacteria: current horizons.

Application of proteomics has made a profound impact on the cyanobacterial research. It has not only provided a global identification of expressed proteins in cyanobacterial cells, but has also brought valuable insights into dynamics of cell responses to environmental challenges, regulation mechanisms, structure of protein complexes, compartmentalization, and other important biological questions. In this review, we highlight current trends in proteomics of cyanobacteria and bring to focus rising techniques which have a huge potential in expanding our knowledge about cyanobacterial proteins and in developing cyanobacteria-based biotechnological applications.

Interplay of SpkG kinase and the Slr0151 protein in the phosphorylation of ferredoxin 5 in Synechocystis sp. strain PCC 6803.

In Synechocystis 6803, the ferredoxin 5 (Fd5) phosphoprotein and the S/T protein kinase SpkG are encoded by the slr0148 and slr0152 genes, respectively, which belong to the slr0144-slr0152 cluster. Using a targeted proteomic approach, we showed that SpkG is responsible for the phosphorylation of Fd5 on residues T18 and T72. Sequence alignments and Fd5 structure modelling suggest that these phosphorylation events modulate protein-protein interaction. Furthermore, Fd5 phosphorylation is affected by the Slr0151 protein encoded by the gene preceding spkG in the gene cluster. We propose that Slr0151 functions as an auxiliary protein in the regulation of the ratio between phosphorylated and nonphosphorylated forms of Fd5.

Septal protein SepJ from the heterocyst-forming cyanobacterium Anabaena forms multimers and interacts with peptidoglycan.

Heterocyst-forming cyanobacteria grow as filaments that can be hundreds of cells long. Proteinaceous septal junctions provide cell-cell binding and communication functions in the filament. In Anabaena sp. strain PCC 7120, the SepJ protein is important for the formation of septal junctions. SepJ consists of integral membrane and extramembrane sections - the latter including linker and coiled-coil domains. SepJ (predicted MW, 81.3 kDa) solubilized from Anabaena membranes was found in complexes of about 296-334 kDa, suggesting that SepJ forms multimeric complexes. We constructed an Anabaena strain producing a double-tagged SepJ protein (SepJ-GFP-His10) and isolated the tagged protein by a two-step affinity chromatography procedure. Analysis of the purified protein preparation provided no indication of the presence of specific SepJ partners, but suggested that SepJ is processed to remove an N-terminal fragment. Additionally, pull-down experiments showed that His6-tagged versions of SepJ and of the SepJ coiled-coil domain interact with Anabaena peptidoglycan (PG). Our results indicate that SepJ forms multimers, that it interacts with PG, and that the coiled-coil domain is involved in this interaction. These observations support the idea that SepJ is a component of the septal junctions that join the cells in the Anabaena filament.

Role of Type 2 NAD(P)H Dehydrogenase NdbC in Redox Regulation of Carbon Allocation in Synechocystis.

NAD(P)H dehydrogenases comprise type 1 (NDH-1) and type 2 (NDH-2s) enzymes. Even though the NDH-1 complex is a well-characterized protein complex in the thylakoid membrane of Synechocystis sp. PCC 6803 (hereafter Synechocystis), the exact roles of different NDH-2s remain poorly understood. To elucidate this question, we studied the function of NdbC, one of the three NDH-2s in Synechocystis, by constructing a deletion mutant (ΔndbC) for a corresponding protein and submitting the mutant to physiological and biochemical characterization as well as to comprehensive proteomics analysis. We demonstrate that the deletion of NdbC, localized to the plasma membrane, affects several metabolic pathways in Synechocystis in autotrophic growth conditions without prominent effects on photosynthesis. Foremost, the deletion of NdbC leads, directly or indirectly, to compromised sugar catabolism, to glycogen accumulation, and to distorted cell division. Deficiencies in several sugar catabolic routes were supported by severe retardation of growth of the ΔndbC mutant under light-activated heterotrophic growth conditions but not under mixotrophy. Thus, NdbC has a significant function in regulating carbon allocation between storage and the biosynthesis pathways. In addition, the deletion of NdbC increases the amount of cyclic electron transfer, possibly via the NDH-12 complex, and decreases the expression of several transporters in ambient CO2 growth conditions.

Dissecting the Photoprotective Mechanism Encoded by the flv4-2 Operon: a Distinct Contribution of Sll0218 in Photosystem II Stabilization.

In Synechocystis sp. PCC 6803, the flv4-2 operon encodes the flavodiiron proteins Flv2 and Flv4 together with a small protein, Sll0218, providing photoprotection for Photosystem II (PSII). Here, the distinct roles of Flv2/Flv4 and Sll0218 were addressed, using a number of flv4-2 operon mutants. In the ∆sll0218 mutant, the presence of Flv2/Flv4 rescued PSII functionality as compared with ∆sll0218-flv2, where neither Sll0218 nor the Flv2/Flv4 heterodimer are expressed. Nevertheless, both the ∆sll0218 and ∆sll0218-flv2 mutants demonstrated deficiency in accumulation of PSII proteins suggesting a role for Sll0218 in PSII stabilization, which was further supported by photoinhibition experiments. Moreover, the accumulation of PSII assembly intermediates occurred in Sll0218-lacking mutants. The YFP-tagged Sll0218 protein localized in a few spots per cell at the external side of the thylakoid membrane, and biochemical membrane fractionation revealed clear enrichment of Sll0218 in the PratA-defined membranes, where the early biogenesis steps of PSII occur. Further, the characteristic antenna uncoupling feature of the ∆flv4-2 operon mutants is shown to be related to PSII destabilization in the absence of Sll0218. It is concluded that the Flv2/Flv4 heterodimer supports PSII functionality, while the Sll0218 protein assists PSII assembly and stabilization, including optimization of light harvesting.

Study of O-Phosphorylation Sites in Proteins Involved in Photosynthesis-Related Processes in Synechocystis sp. Strain PCC 6803: Application of the SRM Approach.

O-Phosphorylation has been shown in photosynthesis-related proteins in a cyanobacterium Synechocystis sp. strain PCC 6803 (thereafter Synechocystis 6803), suggesting that phosphorylation of S, T, and Y residues might be important in photosynthesis-related processes. Investigation of biological roles of these phosphorylation events requires confident knowledge of the phosphorylated sites and prospects for their individual assessment. We performed phosphoproteomic analysis of Synechocystis 6803 using TiO2 enrichment of the phosphopeptides, followed by LC-MS/MS, and discovered 367 phosphorylation sites in 190 proteins participating in various cellular functions. Furthermore, we focused on the large group of phosphoproteins that are involved in light harvesting, photosynthesis-driven electron flow, photoprotection, and CO2 fixation. The SRM approach was applied to verify/improve assignments of phosphorylation sites in these proteins and to investigate possibilities for analysis of phosphopeptide isomers. The SRM assays were designed for peptides comprising 45 phosphorylation sites. The assays contain peptide iRT values and Q1/Q3 transitions comprising those discriminating between phosphopeptide isoforms. The majority of investigated phosphopeptides and phosphorylated isoforms could be individually assessed with the SRM technique. The assays could be potentially used in future quantitative studies to evaluate an extent of phosphorylation in photosynthesis-related proteins in Synechocystis 6803 cells challenged with various environmental stresses.

The NDH-1L-PSI Supercomplex Is Important for Efficient Cyclic Electron Transport in Cyanobacteria.

Two mutants isolated from a tagging library of Synechocystis sp. strain PCC 6803 were sensitive to high light and had a tag in sll1471 encoding CpcG2, a linker protein for photosystem I (PSI)-specific antenna. Both mutants demonstrated strongly impaired NDH-1-dependent cyclic electron transport. Blue native-polyacrylamide gel electrophoresis followed by immunoblotting and mass spectrometry analyses of the wild type and a mutant containing CpcG2 fused with yellow fluorescent protein-histidine6 indicated the presence of a novel NDH-1L-CpcG2-PSI supercomplex, which was absent in the cpcG2 deletion mutant, the PSI-less mutant, and several other strains deficient in NDH-1L and/or NDH-1M. Coimmunoprecipitation and pull-down analyses on CpcG2-yellow fluorescent protein-histidine6, using antibody against green fluorescent protein and nickel column chromatography, confirmed the association of CpcG2 with the supercomplex. Conversely, the use of antibodies against NdhH or NdhK after blue native-polyacrylamide gel electrophoresis and in coimmunoprecipitation experiments verified the necessity of CpcG2 in stabilizing the supercomplex. Furthermore, deletion of CpcG2 destabilized NDH-1L as well as its degradation product NDH-1M and significantly decreased the number of functional PSI centers, consistent with the involvement of CpcG2 in NDH-1-dependent cyclic electron transport. The CpcG2 deletion, however, had no effect on respiration. Thus, we propose that the formation of an NDH-1L-CpcG2-PSI supercomplex in cyanobacteria facilitates PSI cyclic electron transport via NDH-1L.

Changes in Relative Thylakoid Protein Abundance Induced by Fluctuating Light in the Diatom Thalassiosira pseudonana.

One of the hallmarks of marine diatom biology is their ability to cope with rapid changes in light availability due to mixing of the water column and the lens effect. We investigated how irradiance fluctuations influence the relative abundance of key photosynthetic proteins in the centric diatom Thalassiosira pseudonana by means of mass-spectrometry-based approaches for relative protein quantitation. Most notably, fluctuating-light conditions lead to a substantial overall up-regulation of light-harvesting complex proteins as well as several subunits of photosystems II and I. Despite an initial delay in growth under FL, there were no indications of FL-induced photosynthesis limitation, in contrast to other photosynthetic organisms. Our findings further strengthen the notion that diatoms use a qualitatively different mechanism of photosynthetic regulation in which chloroplast-mitochondria interaction has overtaken crucial regulatory processes of photosynthetic light reactions that are typical for the survival of land plants, green algae, and cyanobacteria.

Calcium impacts carbon and nitrogen balance in the filamentous cyanobacterium Anabaena sp. PCC 7120.

Calcium is integral to the perception, communication and adjustment of cellular responses to environmental changes. However, the role of Ca(2+) in fine-tuning cellular responses of wild-type cyanobacteria under favourable growth conditions has not been examined. In this study, extracellular Ca(2+) has been altered, and changes in the whole transcriptome of Anabaena sp. PCC 7120 have been evaluated under conditions replete of carbon and combined nitrogen. Ca(2+) induced differential expression of many genes driving primary cellular metabolism, with transcriptional regulation of carbon- and nitrogen-related processes responding with opposing trends. However, physiological effects of these transcriptional responses on biomass accumulation, biomass composition, and photosynthetic activity over the 24h period following Ca(2+) adjustment were found to be minor. It is well known that intracellular carbon:nitrogen balance is integral to optimal cell growth and that Ca(2+) plays an important role in the response of heterocystous cyanobacteria to combined-nitrogen deprivation. This work adds to the current knowledge by demonstrating a signalling role of Ca(2+) for making sensitive transcriptional adjustments required for optimal growth under non-limiting conditions.

The Flavodiiron Protein Flv3 Functions as a Homo-Oligomer During Stress Acclimation and is Distinct from the Flv1/Flv3 Hetero-Oligomer Specific to the O2 Photoreduction Pathway.

The flavodiiron proteins (FDPs) Flv1 and Flv3 in cyanobacteria function in photoreduction of O2 to H2O, without concomitant formation of reactive oxygen species, known as the Mehler-like reaction. Both Flv1 and Flv3 are essential for growth under fluctuating light (FL) intensities, providing protection for PSI. Here we compared the global transcript profiles of the wild type (WT), Δflv1 and Δflv1/Δflv3 grown under constant light (GL) and FL. In the WT, FL induced the largest down-regulation in transcripts involved in carbon-concentrating mechanisms (CCMs), while those of the nitrogen assimilation pathways increased as compared with GL. Already under GL the Δflv1/Δflv3 double mutant demonstrated a partial down-regulation of transcripts for CCM and nitrogen metabolism, while in FL conditions the transcripts for nitrogen assimilation were strongly down-regulated. Many alterations were specific only for Δflv1/Δflv3, and not detected in Δflv1, suggesting that certain transcripts are affected primarily because of the lack of flv3 By constructing the strains overproducing solely either Flv1 or Flv3, we demonstrate that the homo-oligomers of these proteins also function in acclimation of cells to FL, by catalyzing reactions with as yet unidentified components, while the presence of both Flv1 and Flv3 is a prerequisite for the Mehler-like reaction and thus the electron transfer to O2 Considering the low expression of flv1, it is unlikely that the Flv1 homo-oligomer is present in the WT.

Integration of photosynthesis, development and stress as an opportunity for plant biology.

With the tremendous progress of the past decades, molecular plant science is becoming more unified than ever. We now have the exciting opportunity to further connect subdisciplines and understand plants as whole organisms, as will be required to efficiently utilize them in natural and agricultural systems to meet human needs. The subfields of photosynthesis, plant developmental biology and plant stress are used as examples to discuss how plant science can become better integrated. The challenges, strategies and rich opportunities for the integration of the plant sciences are discussed. In recent years, more and more overlap between various subdisciplines has been inadvertently discovered including tradeoffs that may occur in plants engineered for biotechnological applications. Already important, bioinformatics and computational modelling will become even more central to structuring and understanding the ever growing amounts of data. The process of integrating and overlapping fields in plant biology research is advancing, but plant science will benefit from dedicating more effort and urgency to reach across its boundaries.

Chlamydomonas Flavodiiron Proteins Facilitate Acclimation to Anoxia During Sulfur Deprivation.

The flavodiiron proteins (FDPs) are involved in the detoxification of oxidative compounds, such as nitric oxide (NO) or O(2) in Archaea and Bacteria. In cyanobacteria, the FDPs Flv1 and Flv3 are essential in the light-dependent reduction of O(2) downstream of PSI. Phylogenetic analysis revealed that two genes (flvA and flvB) in the genome of Chlamydomonas reinhardtii show high homology to flv1 and flv3 genes of the cyanobacterium Synechocystis sp. PCC 6803. The physiological role of these FDPs in eukaryotic green algae is not known, but it is of a special interest since these phototrophic organisms perform oxygenic photosynthesis similar to higher plants, which do not possess FDP homologs. We have analyzed the levels of flvA and flvB transcripts in C. reinhardtii cells under various environmental conditions and showed that these genes are highly expressed under ambient CO(2) levels and during the early phase of acclimation to sulfur deprivation, just before the onset of anaerobiosis and the induction of efficient H(2) photoproduction. Importantly, the increase in transcript levels of the flvA and flvB genes was also corroborated by protein levels. These results strongly suggest the involvement of FLVA and FLVB proteins in alternative electron transport.

Transcriptomic and Proteomic Profiling of Anabaena sp. Strain 90 under Inorganic Phosphorus Stress.

Inorganic phosphorus (Pi) is one of the main growth-limiting factors of diazotrophic cyanobacteria. Due to human activity, the availability of Pi has increased in water bodies, resulting in eutrophication and the formation of massive cyanobacterial blooms. In this study, we examined the molecular responses of the cyanobacterium Anabaena sp. strain 90 to phosphorus deprivation, aiming at the identification of candidate genes to monitor the Pi status in cyanobacteria. Furthermore, this study increased the basic understanding of how phosphorus affects diazotrophic and bloom-forming cyanobacteria as a major growth-limiting factor. Based on RNA sequencing data, we identified 246 differentially expressed genes after phosphorus starvation and 823 differentially expressed genes after prolonged Pi limitation, most of them related to central metabolism and cellular growth. The transcripts of the genes related to phosphorus transport and assimilation (pho regulon) were most upregulated during phosphorus depletion. One of the most increased transcripts encodes a giant protein of 1,869 amino acid residues, which contains, among others, a phytase-like domain. Our findings predict its crucial role in phosphorus starvation, but future studies are still needed. Using two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we found 43 proteins that were differentially expressed after prolonged phosphorus stress. However, correlation analysis unraveled an association only to some extent between the transcriptomic and proteomic abundances. Based on the present results, we suggest that the method used for monitoring the Pi status in cyanobacterial bloom should contain wider combinations of pho regulon genes (e.g., PstABCS transport systems) in addition to the commonly used alkaline phosphatase gene alone.

Draft genome sequence of calothrix strain 336/3, a novel h2-producing cyanobacterium isolated from a finnish lake.

We announce the draft genome sequence of Calothrix strain 336/3, an N2-fixing heterocystous filamentous cyanobacterium isolated from a natural habitat. Calothrix 336/3 produces higher levels of hydrogen than Nostoc punctiforme PCC 73102 and Anabaena strain PCC 7120 and, therefore, is of interest for potential technological applications.

Proteomic approaches in research of cyanobacterial photosynthesis.

Oxygenic photosynthesis in cyanobacteria, algae, and plants is carried out by a fabulous pigment-protein machinery that is amazingly complicated in structure and function. Many different approaches have been undertaken to characterize the most important aspects of photosynthesis, and proteomics has become the essential component in this research. Here we describe various methods which have been used in proteomic research of cyanobacteria, and demonstrate how proteomics is implemented into on-going studies of photosynthesis in cyanobacterial cells.

SASP, a Senescence-Associated Subtilisin Protease, is involved in reproductive development and determination of silique number in Arabidopsis.

Senescence involves increased expression of proteases, which may participate in nitrogen recycling or cellular signalling. 2D zymograms detected two protein species with increased proteolytic activity in senescing leaves of Arabidopsis thaliana. A proteomic analysis revealed that both protein species correspond to a subtilisin protease encoded by At3g14067, termed Senescence-Associated Subtilisin Protease (SASP). SASP mRNA levels and enzyme activity increase during leaf senescence in leaves senescing during both the vegetative or the reproductive phase of the plant life cycle, but this increase is more pronounced in reproductive plants. SASP is expressed in all above-ground organs, but not in roots. Putative AtSASP orthologues were identified in dicot and monocot crop species. A phylogenetic analysis shows AtSASP and its putative orthologues clustering in one discrete group of subtilisin proteases in which no other Arabidospsis subtilisin protease is present. Phenotypic analysis of two knockout lines for SASP showed that mutant plants develop more inflorescence branches during reproductive development. Both AtSASP and its putative rice orthologue (OsSASP) were constitutively expressed in sasp-1 to complement the mutant phenotype. At maturity, sasp-1 plants produced 25% more inflorescence branches and siliques than either the wild-type or the rescued lines. These differences were mostly due to an increased number of second and third order branches. The increased number of siliques was compensated for by a small decrease (5.0%) in seed size. SASP downregulates branching and silique production during monocarpic senescence, and its function is at least partially conserved between Arabidopsis and rice.

Posttranslational modifications of FERREDOXIN-NADP+ OXIDOREDUCTASE in Arabidopsis chloroplasts.

Rapid responses of chloroplast metabolism and adjustments to photosynthetic machinery are of utmost importance for plants' survival in a fluctuating environment. These changes may be achieved through posttranslational modifications of proteins, which are known to affect the activity, interactions, and localization of proteins. Recent studies have accumulated evidence about the crucial role of a multitude of modifications, including acetylation, methylation, and glycosylation, in the regulation of chloroplast proteins. Both of the Arabidopsis (Arabidopsis thaliana) leaf-type FERREDOXIN-NADP(+) OXIDOREDUCTASE (FNR) isoforms, the key enzymes linking the light reactions of photosynthesis to carbon assimilation, exist as two distinct forms with different isoelectric points. We show that both AtFNR isoforms contain multiple alternative amino termini and undergo light-responsive addition of an acetyl group to the α-amino group of the amino-terminal amino acid of proteins, which causes the change in isoelectric point. Both isoforms were also found to contain acetylation of a conserved lysine residue near the active site, while no evidence for in vivo phosphorylation or glycosylation was detected. The dynamic, multilayer regulation of AtFNR exemplifies the complex regulatory network systems controlling chloroplast proteins by a range of posttranslational modifications, which continues to emerge as a novel area within photosynthesis research.