Photosystem II (PSII) is the multi-subunit light-driven oxidoreductase that drives photosynthetic electron transport using electrons extracted from water. To investigate the initial steps of PSII ...assembly, we used strains of the cyanobacterium Synechocystis sp. PCC 6803 arrested at early stages of PSII biogenesis and expressing affinity-tagged PSII subunits to isolate PSII reaction center assembly (RCII) complexes and their precursor D1 and D2 modules (D1mod and D2mod). RCII preparations isolated using either a His-tagged D2 or a FLAG-tagged PsbI subunit contained the previously described RCIIa and RCII* complexes that differ with respect to the presence of the Ycf39 assembly factor and high light-inducible proteins (Hlips) and a larger complex consisting of RCIIa bound to monomeric PSI. All RCII complexes contained the PSII subunits D1, D2, PsbI, PsbE, and PsbF and the assembly factors rubredoxin A and Ycf48, but we also detected PsbN, Slr1470, and the Slr0575 proteins, which all have plant homologs. The RCII preparations also contained prohibitins/stomatins (Phbs) of unknown function and FtsH protease subunits. RCII complexes were active in light-induced primary charge separation and bound chlorophylls (Chls), pheophytins, beta-carotenes, and heme. The isolated D1mod consisted of D1/PsbI/Ycf48 with some Ycf39 and Phb3, while D2mod contained D2/cytochrome b559 with co-purifying PsbY, Phb1, Phb3, FtsH2/FtsH3, CyanoP, and Slr1470. As stably bound, Chl was detected in D1mod but not D2mod, formation of RCII appears to be important for stable binding of most of the Chls and both pheophytins. We suggest that Chl can be delivered to RCII from either monomeric Photosystem I or Ycf39/Hlips complexes.
Oxygenic photosynthesis relies on accessory factors to promote the assembly and maintenance of the photosynthetic apparatus in the thylakoid membranes. The highly conserved membrane-bound ...rubredoxin-like protein RubA has previously been implicated in the accumulation of both PSI and PSII, but its mode of action remains unclear. Here, we show that RubA in the cyanobacterium
sp PCC 6803 is required for photoautotrophic growth in fluctuating light and acts early in PSII biogenesis by promoting the formation of the heterodimeric D1/D2 reaction center complex, the site of primary photochemistry. We find that RubA, like the accessory factor Ycf48, is a component of the initial D1 assembly module as well as larger PSII assembly intermediates and that the redox-responsive rubredoxin-like domain is located on the cytoplasmic surface of PSII complexes. Fusion of RubA to Ycf48 still permits normal PSII assembly, suggesting a spatiotemporal proximity of both proteins during their action. RubA is also important for the accumulation of PSI, but this is an indirect effect stemming from the downregulation of light-dependent chlorophyll biosynthesis induced by PSII deficiency. Overall, our data support the involvement of RubA in the redox control of PSII biogenesis.
The negatively charged lipid phosphatidylglycerol (PG) constitutes up to 10% of total lipids in photosynthetic membranes, and its deprivation in cyanobacteria is accompanied by chlorophyll (Chl) ...depletion. Indeed, radioactive labeling of the PG-depleted ΔpgsA mutant of Synechocystis sp. strain PCC 6803, which is not able to synthesize PG, proved the inhibition of Chl biosynthesis caused by restriction on the formation of 5-aminolevulinic acid and protochlorophyllide. Although the mutant accumulated chlorophyllide, the last Chl precursor, we showed that it originated from dephytylation of existing Chl and not from the block in the Chl biosynthesis. The lack of de novo-produced Chl under PG depletion was accompanied by a significantly weakened biosynthesis of both monomeric and trimeric photosystem I (PSI) complexes, although the decrease in cellular content was manifested only for the trimeric form. However, our analysis of ΔpgsA mutant, which lacked trimeric PSI because of the absence of the PsaL subunit, suggested that the virtual stability of monomeric PSI is a result of disintegration of PSI trimers. Interestingly, the loss of trimeric PSI was accompanied by accumulation of monomeric PSI associated with the newly synthesized CP43 subunit of photosystem II. We conclude that the absence of PG results in the inhibition of Chl biosynthetic pathway, which impairs synthesis of PSI, despite the accumulation of chlorophyllide released from the degraded Chl proteins. Based on the knowledge about the role of PG in prokaryotes, we hypothesize that the synthesis of Chl and PSI complexes are colocated in a membrane microdomain requiring PG for integrity.
To study the regulation of carotenoid biosynthesis and accumulation, the microalga Scenedesmus sp. might represent an alternative model microorganism to the well established Haematococcus pluvialis ...or Dunaliella salina. Scenedesmus sp. strain CCALA 1074 can produce high levels of carotenoids, mainly lutein, over all growth phases. In exponentially growing cultures, total carotenoids and lutein accumulated to levels of up to 1 and 0.75% DW, respectively. In early stationary phase, total carotenoids increased to 2.3% DW; lutein however remained unchanged and additional minor ketocarotenoids such as astaxanthin, echinenone and a number of unidentified carotenoid species were also present. Nitrate-limited cultures were supplemented either with nitrate or complete growth medium in early stationary phase and were cultivated either in light or in darkness. As expected, in light, cells started to divide and cultures grew; surprisingly however, in darkness, cell division was blocked after supplementation, independent of the nutrient added. Carotenoid synthesis also only proceeded in the light, whereas, in darkness, carotenoid content remained constant. When cultivated in an outdoor thin-layer photobioreactor (PBR), the greatest increase in carotenoid accumulation occurred under conditions of nitrogen sufficiency and high light. These results suggest that in Scenedesmus sp., light is a critical factor in the accumulation of carotenoids, and nitrogen availability plays only a minor role.
Ferrochelatase (FeCh) is an essential enzyme catalyzing the synthesis of heme. Interestingly, in cyanobacteria, algae, and plants, FeCh possesses a conserved transmembrane chlorophyll a/b binding ...(CAB) domain that resembles the first and the third helix of light-harvesting complexes, including a chlorophyll-binding motif. Whether the FeCh CAB domain also binds chlorophyll is unknown. Here, using biochemical and radiolabeled precursor experiments, we found that partially inhibited activity of FeCh in the cyanobacterium Synechocystis PCC 6803 leads to overproduction of chlorophyll molecules that accumulate in the thylakoid membrane and, together with carotenoids, bind to FeCh. We observed that pigments bound to purified FeCh are organized in an energy-dissipative conformation and further show that FeCh can exist in vivo as a monomer or a dimer depending on its own activity. However, pigmented FeCh was purified exclusively as a dimer. Separately expressed and purified FeCH CAB domain contained a pigment composition similar to that of full-length FeCh and retained its quenching properties. Phylogenetic analysis suggested that the CAB domain was acquired by a fusion between FeCh and a single-helix, high light-inducible protein early in the evolution of cyanobacteria. Following this fusion, the FeCh CAB domain with a functional chlorophyll-binding motif was retained in all currently known cyanobacterial genomes except for a single lineage of endosymbiotic cyanobacteria. Our findings indicate that FeCh from Synechocystis exists mostly as a pigment-free monomer in cells but can dimerize, in which case its CAB domain creates a functional pigment-binding segment organized in an energy-dissipating configuration.
Macromolecular membrane assemblies of chlorophyll-protein complexes efficiently harvest and trap light energy for photosynthesis. To investigate the delivery of chlorophylls to the newly synthesized ...photosystem apoproteins, a terminal enzyme of chlorophyll biosynthesis, chlorophyll synthase (ChIG), was tagged in the cyanobacterium Synechocystis PCC 6803 (Synechocystis) and used as bait in pull-down experiments. We retrieved an enzymatically active complex comprising ChIG and the high-light-inducible protein HliD, which associates with the Ycf39 protein, a putative assembly factor for photosystem II, and with the YidC/Alb3 insertase. 2D electrophoresis and immunoblotting also provided evidence for the presence of SecY and ribosome subunits. The isolated complex contained chlorophyll, chlorophyllide, and carotenoid pigments. Deletion of hliD elevated the level of the ChIG substrate, chlorophyllide, more than 6-fold; HliD is apparently required for assembly of FLAGChlG into larger complexes with other proteins such as Ycf39. These data reveal a link between chlorophyll biosynthesis and the Sec/YidC-dependent cotranslational insertion of nascent photosystem polypeptides into membranes. We expect that this close physical linkage coordinates the arrival of pigments and nascent apoproteins to produce photosynthetic pigmentprotein complexes with minimal risk of accumulating phototoxic unbound chlorophylls.
Haptoglobin is a plasma protein of mammals that plays a crucial role in vascular homeostasis by binding free haemoglobin released from ruptured red blood cells. Trypanosoma brucei can exploit this by ...internalising haptoglobin-haemoglobin complex to acquire host haem. Here, we investigated the impact of haptoglobin deficiency (Hp-/-) on T. brucei brucei infection and the parasite´s capacity to internalise haemoglobin in a Hp-/- mouse model. The infected Hp-/- mice exhibited normal disease progression, with minimal weight loss and no apparent organ pathology, similarly to control mice. While the proteomic profile of mouse sera significantly changed in response to T. b. brucei , no differences in the infection response markers of blood plasma between Hp-/- and control Black mice were observed. Similarly, very few quantitative differences were observed between the proteomes of parasites harvested from Hp-/- and Black mice, including both endogenous proteins and internalised host proteins. While haptoglobin was indeed absent from parasites isolated from Hp-/-mice, haemoglobin peptides were unexpectedly detected in parasites from both Hp-/- and Black mice. Combined, the data support the dispensability of haptoglobin for haemoglobin internalisation by T. b. brucei during infection in mice. Since the trypanosomes knock-outs for their haptoglobin-haemoglobin receptor (HpHbR) internalised significantly less haemoglobin from Hp-/- mice compared to those isolated from Black mice, it suggests that T. b. brucei employs also an HpHbR-independent haptoglobin-mediated mode for haemoglobin internalisation. Our study reveals a so-far hidden flexibility of haemoglobin acquisition by T. b. brucei and offers novel insights into alternative haemoglobin uptake pathways.
In natural environments, photosynthetic organisms adjust their metabolism to cope with the fluctuating availability of combined nitrogen sources, a growth-limiting factor. For acclimation, the ...dynamic degradation/synthesis of tetrapyrrolic pigments, as well as of the amino acid arginine, is pivotal; however, there has been no evidence that these processes could be functionally coupled. Using co-immunopurification and spectral shift assays, we found that in the cyanobacterium Synechocystis sp. PCC 6803, the arginine metabolism-related ArgD and CphB enzymes form protein complexes with Gun4, an essential protein for chlorophyll biosynthesis. Gun4 binds ArgD with high affinity, and the Gun4-ArgD complex accumulates in cells supplemented with ornithine, a key intermediate of the arginine pathway. Elevated ornithine levels restricted de novo synthesis of tetrapyrroles, which arrested the recovery from nitrogen deficiency. Our data reveal a direct crosstalk between tetrapyrrole biosynthesis and arginine metabolism that highlights the importance of balancing photosynthetic pigment synthesis with nitrogen homeostasis.
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•ArgD of arginine pathway interacts with the Gun4 protein of chlorophyll biosynthesis•Ornithine stimulates the ArgD-Gun4 interaction and inhibits tetrapyrrole synthesis•The inhibition by ornithine is released in a strain lacking the ArgD-Gun4 complex
Kiss et al. demonstrate that the synthesis of the main photosynthetic pigment, chlorophyll, and the metabolism of arginine (managing N-stockpile) are functionally coupled via protein-protein interactions in cyanobacteria.
Increasing crop productivity under optimal conditions and mitigating yield losses under stressful conditions is a major challenge in contemporary agriculture. We have recently identified an effective ...anti-senescence compound (MTU, 1-(2-methoxyethyl)-3-(1,2,3-thiadiazol-5yl)urea) in
studies. Here, we show that MTU delayed both age- and stress-induced senescence of wheat plants (
L.) by enhancing the abundance of PSI supercomplex with LHCa antennae (PSI-LHCa) and promoting the cyclic electron flow (CEF) around PSI. We suppose that this rarely-observed phenomenon blocks the disintegration of photosynthetic apparatus and maintains its activity as was reflected by the faster growth rate of wheat in optimal conditions and under drought and heat stress. Our multiyear field trial analysis further shows that the treatment with 0.4 g ha
of MTU enhanced average grain yields of field-grown wheat and barley (
L.) by 5-8%. Interestingly, the analysis of gene expression and hormone profiling confirms that MTU acts without the involvement of cytokinins or other phytohormones. Moreover, MTU appears to be the only chemical reported to date to affect PSI stability and activity. Our results indicate a central role of PSI and CEF in the onset of senescence with implications in yield management at least for cereal species.
Stress-induced senescence is a global agro-economic problem. Cytokinins are considered to be key plant anti-senescence hormones, but despite this practical function their use in agriculture is ...limited because cytokinins also inhibit root growth and development. We explored new cytokinin analogs by synthesizing a series of 1,2,3-thiadiazol-5-yl urea derivatives. The most potent compound, 1-(2-methoxy-ethyl)-3-1,2,3-thiadiazol-5-yl urea (ASES - Anti-Senescence Substance), strongly inhibited dark-induced senescence in leaves of wheat (
L.) and
. The inhibitory effect of ASES on wheat leaf senescence was, to the best of our knowledge, the strongest of any known natural or synthetic compound.
, ASES also improved the salt tolerance of young wheat plants. Interestingly, ASES did not affect root development of wheat and Arabidopsis, and molecular and classical cytokinin bioassays demonstrated that ASES exhibits very low cytokinin activity. A proteomic analysis of the ASES-treated leaves further revealed that the senescence-induced degradation of photosystem II had been very effectively blocked. Taken together, our results including data from cytokinin content analysis demonstrate that ASES delays leaf senescence by mechanism(s) different from those of cytokinins and, more effectively. No such substance has yet been described in the literature, which makes ASES an interesting tool for research of photosynthesis regulation. Its simple synthesis and high efficiency predetermine ASES to become also a potent plant stress protectant in biotechnology and agricultural industries.
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