Site of inhibition by 2-amino-1,1,3-tricyanopropene of photosynthetic oxygen evolution by spinach leaf chloroplasts (Short Communication).
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ABSTRACT:
SUBMITTER: Burden EM
PROVIDER: S-EPMC1177857 | biostudies-other | 1973 Jun
REPOSITORIES: biostudies-other
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Project description:The light-driven splitting of water to oxygen (O2) is catalyzed by a protein-bound tetra-manganese penta-oxygen calcium (Mn4O5Ca) cluster in Photosystem II. In the current study, we used a large-scale integration (LSI)-based amperometric sensor array system, designated Bio-LSI, to perform two-dimensional imaging of light-induced O2 evolution from spinach leaves. The employed Bio-LSI chip consists of 400 sensor electrodes with a pitch of 250 μm for fast electrochemical imaging. Spinach leaves were illuminated to varying intensities of white light (400-700 nm) which induced oxygen evolution and subsequent electrochemical images were collected using the Bio-LSI chip. Bio-LSI images clearly showed the dose-dependent effects of the light-induced oxygen release from spinach leaves which was then significantly suppressed in the presence of urea-type herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Our results clearly suggest that light-induced oxygen evolution can be monitored using the chip and suggesting that the Bio-LSI is a promising tool for real-time imaging. To the best of our knowledge, this report is the first to describe electrochemical imaging of light-induced O2 evolution using LSI-based amperometric sensors in plants.
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Project description:Diclofenac, often detected in environmental samples, poses a potential hazard to the aquatic environment. The present study aimed to understand the effect of this drug on photosynthetic apparatus, which is a little-known aspect of its phytotoxicity. Chloroplasts and thylakoids isolated from spinach (Spinacia oleracea) were used for this study and treated with various concentrations of diclofenac (from 125 to 4000 μM). The parameters of chlorophyll a fluorescence (the OJIP test) as measurements for both the intact chloroplasts and the thylakoid membranes revealed that isolated thylakoids showed greater sensitivity to the drug than chloroplasts. The relatively high concentration of diclofenac that is required to inhibit chloroplast and thylakoid functions suggests a narcotic effect of that drug on photosynthetic membranes, rather than a specific interaction with a particular element of the electron transport chain. Using confocal microscopy, we confirmed the degradation of the chloroplast structure after DCF treatment, which has not been previously reported in the literature. In conclusion, it can be assumed that diclofenac's action originated from a non-specific interaction with photosynthetic membranes, leading to the disruption in the function of the electron transport chain. This, in turn, decreases the efficiency of photosynthesis, transforming part of the PSII reaction centers into heat sinks and enhancing non-photochemical energy dissipation.
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Project description:Land plants encountering low water potentials (low psiw) close their stomata, restricting CO2 entry and potentially photosynthesis. To determine the impact of stomatal closure, photosynthetic O2 evolution was investigated in leaf discs from sunflower (Helianthus annuus L.) plants after removing the lower epidermis at low psiw. Wounding was minimal as evidenced by O2 evolution nearly as rapid as that in intact discs. O2 evolution was maximal in 1% CO2 in the peeled discs and was markedly inhibited when psiw was below -1.1 MPa. CO2 entered readily at all psiw, as demonstrated by varying the CO2 concentration. Results were the same whether the epidermis was removed before or after low psiw was imposed. Due to the lack of an epidermis and ready movement or CO2 through the mesophyll, the loss in O2 evolving activity was attributed entirely to photosynthetic metabolism. Intact leaf discs showed a similar loss in activity when measured at a CO2 concentration of 5%, which supported maximum O2 evolution at low psiw. In 1% CO2, however, O2 evolution at low psiw was below the maximum, presumably because stomatal closure restricted CO2 uptake. The inhibition was larger than in peeled discs at psiw between -1 and -1.5 MPa but became the same as in peeled discs at lower psiw. Therefore. as photosynthesis began to be inhibited by metabolism at low psiw, stomatal closure added to the inhibition. As psiw became more negative, the inhibition became entirely metabolic.
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Project description:Photosynthetic oxygen evolution is performed at the Mn cluster in photosystem II (PSII). The advent of this reaction on ancient Earth changed its environment by generating an oxygenic atmosphere. However, how oxygen evolution originated during the PSII evolution remains unknown. Here, we characterize the site-directed mutants at the carboxylate ligands to the Mn cluster in cyanobacterial PSII. A His residue replaced for D1-D170 is found to be post-translationally converted to the original Asp to recover oxygen evolution. Gln/Asn residues in the mutants at D1-E189/D1-D342 are also converted to Glu/Asp, suggesting that amino-acid conversion is a common phenomenon at the ligand sites of the Mn cluster. We hypothesize that post-translational generation of carboxylate ligands in ancestral PSII could have led to the formation of a primitive form of the Mn cluster capable of partial water oxidation, which could have played a crucial role in the evolutionary process of photosynthetic oxygen evolution.
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Project description:Spinach chloroplasts catalyse the incorporation of Fe(2+) into protoporphyrin, mesoporphyrin and deuteroporphyrin to form the corresponding haems. This ferrochelatase activity was detected by pyridine haemochrome formation with acetone-dried powders of chloroplasts, or from the formation of [(59)Fe]haems by intact chloroplasts. Decreasing the mitochondrial contamination of the chloroplasts by density-gradient centrifugation did not cause any loss of activity: spinach ferrochelatase appears to be principally a chloroplast enzyme. The characteristics of the enzyme were examined by using [(59)Fe]haem assay. The activity was pH-dependent: for both mesohaem and protohaem formation there were two pH maxima, a major peak at about pH7.8 and a smaller peak at about pH9.2. Lineweaver-Burk plots showed that the K(m) for Fe(2+) incorporation into protoporphyrin was 8mum and that for Fe(2+) incorporation into mesoporphyrin was 36mum. At non-saturating Fe(2+) concentrations the K(m) for protoporphyrin was 0.2mum and that for mesoporphyrin was 0.4mum. Ferrochelatase was not solubilized by treatment of chloroplasts with ultrasound but was solubilized by stirring in 1% (w/v) Tween 20 at pH10.4. Unlike the rat liver mitochondrial enzyme, chloroplast ferrochelatase was not stimulated by treatment with selected organic solvents. The spinach enzyme was inactive in aerobic conditions and it was shown by using an oxygen electrode that under such conditions the addition of Fe(2+) to buffer solutions caused a rapid uptake of dissolved oxygen, believed to be due to the oxidation of Fe(2+) to Fe(3+); Fe(3+) is not a substrate for ferrochelatase.
| S-EPMC1198618 | biostudies-other
Project description:We recently reported [Huber, Huber & Nielsen (1989) Arch. Biochem. Biophys. 270, 681-690] that spinach (Spinacia oleracea L.) sucrose-phosphate synthase (SPS; EC 2.4.1.14) was phosphorylated in vivo when leaves were fed [32P]Pi. In vitro the enzyme was phosphorylated and inactivated by using [gamma-32P]ATP. We now report that SPS is phosphorylated both in vivo and in vitro on serine residues. The protein is phosphorylated at multiple sites both in vivo and in vitro as indicated by two-dimensional peptide maps of the immunopurified SPS protein. After being fed with radiolabel, leaves were illuminated or given mannose (which activates the enzyme), in the presence or absence of okadaic acid. Feeding okadaic acid to leaves decreased the SPS activation state in the dark and light and in leaves fed mannose. Across all the treatments, the activation state of SPS in situ was inversely related to the labelling of two phosphopeptides (designated phosphopeptides 5 and 7). These two phosphopeptides are phosphorylated when SPS is inactivated in vitro with [gamma-32P]ATP, and thus are designated as regulatory (inhibitory) sites [Huber & Huber (1991) Biochim. Biophys. Acta 1091, 393-400]. Okadaic acid increased the total 32P-labelling of SPS and in particular increased labelling of the two regulatory sites, which explains the decline in activation state. In the presence of okadaic acid, two cryptic phosphorylation sites became labelled in vivo that were not apparent in the absence of the inhibitor. Overall, the results suggest that light/dark regulation of SPS activity occurs as a result of regulatory serine phosphorylation. Multiple sites are phosphorylated in vivo, but two sites in particular appear to regulate activity and dephosphorylation of these sites in vivo is sensitive to okadaic acid.
| S-EPMC1130968 | biostudies-other
Project description:1. Photosynthetic electron transport from water to lipophilic Photosystem II acceptors was stimulated 3--5-fold by high concentrations (greater than or equal to 1 M) of salts containing anions such as citrate, succinate and phosphate that are high in the Hofmeister series. 2. In trypsin-treated chloroplasts, K3Fe(CN)6 reduction insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea was strongly stimulated by high concentrations of potassium citrate, but there was much less stimulation of 2,6-dichloroindophenol reduction in Tris-treated chloroplasts supplied with 1,5-diphenylcarbazide as artificial donor. The results suggest that the main site of action of citrate was the O2-evolving complex of Photosystem II. 3. Photosystem I partial reactions were also stimulated by intermediate concentrations of citrate (up to 2-fold stimulation by 0.6--0.8 M-citrate), but were inhibited at the highest concentrations. The observed stimulation may have been caused by stabilizaton of plastocyanin that was complexed with the Photosystem I reaction centre, 4. At 1 M, potassium citrate protected O2 evolution against denaturation by heat or by the chaotropic agent NaNO3. 5. It is suggested that anions high in the Hofmeister series stimulated and stabilized electron transport by enhancing water structure around the protein complexes in the thylakoid membrane.
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Project description:BackgroundChloroplasts descended from cyanobacteria and have a drastically reduced genome following an endosymbiotic event. Many genes of the ancestral cyanobacterial genome have been transferred to the plant nuclear genome by horizontal gene transfer. However, a selective set of metabolism pathways is maintained in chloroplasts using both chloroplast genome encoded and nuclear genome encoded enzymes. As an organelle specialized for carrying out photosynthesis, does the chloroplast metabolic network have properties adapted for higher efficiency of photosynthesis? We compared metabolic network properties of chloroplasts and prokaryotic photosynthetic organisms, mostly cyanobacteria, based on metabolic maps derived from genome data to identify features of chloroplast network properties that are different from cyanobacteria and to analyze possible functional significance of those features.ResultsThe properties of the entire metabolic network and the sub-network that consists of reactions directly connected to the Calvin Cycle have been analyzed using hypergraph representation. Results showed that the whole metabolic networks in chloroplast and cyanobacteria both possess small-world network properties. Although the number of compounds and reactions in chloroplasts is less than that in cyanobacteria, the chloroplast's metabolic network has longer average path length, a larger diameter, and is Calvin Cycle -centered, indicating an overall less-dense network structure with specific and local high density areas in chloroplasts. Moreover, chloroplast metabolic network exhibits a better modular organization than cyanobacterial ones. Enzymes involved in the same metabolic processes tend to cluster into the same module in chloroplasts.ConclusionIn summary, the differences in metabolic network properties may reflect the evolutionary changes during endosymbiosis that led to the improvement of the photosynthesis efficiency in higher plants. Our findings are consistent with the notion that since the light energy absorption, transfer and conversion is highly efficient even in photosynthetic bacteria, the further improvements in photosynthetic efficiency in higher plants may rely on changes in metabolic network properties.
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Project description:Main conclusionThe absence of state transitions in a Nt(Hn) cybrid is due to a cleavage of the threonine residue from the misprocessed N-terminus of the LHCII polypeptides. The cooperation between the nucleus and chloroplast genomes is essential for plant photosynthetic fitness. The rapid and specific interactions between nucleus-encoded and chloroplast-encoded proteins are under intense investigation with potential for applications in agriculture and renewable energy technology. Here, we present a novel model for photosynthesis research in which alien henbane (Hyoscyamus niger) chloroplasts function on the nuclear background of a tobacco (Nicotiana tabacum). The result of this coupling is a cytoplasmic hybrid (cybrid) with inhibited state transitions-a mechanism responsible for balancing energy absorption between photosystems. Protein analysis showed differences in the LHCII composition of the cybrid plants. SDS-PAGE analysis revealed a novel banding pattern in the cybrids with at least one additional 'LHCII' band compared to the wild-type parental species. Proteomic work suggested that the N-terminus of at least some of the cybrid Lhcb proteins was missing. These findings provide a mechanistic explanation for the lack of state transitions-the N-terminal truncation of the Lhcb proteins in the cybrid included the threonine residue that is phosphorylated/dephosphorylated in order to trigger state transitions and therefore crucial energy balancing mechanism in plants.
| S-EPMC6602992 | biostudies-literature
Project description:Main conclusionThe absence of state transitions in a Nt(Hn) cybrid is due to a cleavage of the threonine residue from the misprocessed N-terminus of the LHCII polypeptides. The cooperation between the nucleus and chloroplast genomes is essential for plant photosynthetic fitness. The rapid and specific interactions between nucleus-encoded and chloroplast-encoded proteins are under intense investigation with potential for applications in agriculture and renewable energy technology. Here, we present a novel model for photosynthesis research in which alien henbane (Hyoscyamus niger) chloroplasts function on the nuclear background of a tobacco (Nicotiana tabacum). The result of this coupling is a cytoplasmic hybrid (cybrid) with inhibited state transitions-a mechanism responsible for balancing energy absorption between photosystems. Protein analysis showed differences in the LHCII composition of the cybrid plants. SDS-PAGE analysis revealed a novel banding pattern in the cybrids with at least one additional 'LHCII' band compared to the wild-type parental species. Proteomic work suggested that the N-terminus of at least some of the cybrid Lhcb proteins was missing. These findings provide a mechanistic explanation for the lack of state transitions-the N-terminal truncation of the Lhcb proteins in the cybrid included the threonine residue that is phosphorylated/dephosphorylated in order to trigger state transitions and therefore crucial energy balancing mechanism in plants.
| S-EPMC6602992 | biostudies-literature