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A new enzyme for the interconversion of pyruvate and phosphopyruvate and its role in the C4 dicarboxylic acid pathway of photosynthesis.

1. An enzyme was isolated from leaves of tropical grasses that catalyses the reversible conversion of pyruvate, ATP and orthophosphate into phosphopyruvate, AMP and pyrophosphate. A requirement for Mg(2+) could not be replaced by Mn(2+) or Ca(2+). 2. By replacing orthophosphate with [(32)P]orthophosphate or with arsenate, evidence was provided that the orthophosphate consumed appears in pyrophosphate. 3. Without Mg(2+) or 2-mercaptoethanol the enzyme was rapidly and irreversibly inactivated. EDTA only partially replaced the requirement for the thiol compound. The enzyme was considerably more unstable at 0 degrees or when frozen than at 22 degrees . Even with the best conditions devised the enzyme lost about 25% of its activity every 3hr. 4. The activities of the enzyme in leaves of the tropical grasses sugar cane (Saccharum hybrid var. Pindar), maize (Zea mays) and sorghum (Sorghum vulgare) were comparable with their maximum photosynthesis rates. The enzyme was not detectable in leaf extracts from several other plants. 5. Its role in photosynthesis is discussed.

Adenine Nucleotides↗

Photosynthesis by isolated chloroplasts. Reversal of orthophosphate inhibition by Calvin-cycle intermediates.

1. The orthophosphate inhibition of photosynthesis by isolated spinach chloroplasts can be reversed by 3-phosphoglycerate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, fructose 6-phosphate and fructose 1,6-diphosphate. 2. Metabolically related compounds such as ribulose 1,5-diphosphate, glucose 6-phosphate, 6-phosphogluconate and phosphoenolpyruvate are ineffective. 3. The kinetics of reversal are characteristic of the intermediate used, but, in each instance, the onset of oxygen evolution is accompanied by a carbon dioxide fixation and except with 3-phosphoglycerate the stoicheiometry is close to unity. 4. The nature of orthophosphate inhibition and its reversal is discussed in relation to metabolic control of photosynthesis.

Carbon Dioxide↗

Auxiliary functions in photosynthesis: the role of the FtsH protease.

Oxygenic photosynthesis can be described effectively by using two long-standing models: the Z-scheme and the chemiosmotic hypothesis. However, these models do not reveal the dynamic nature of the thylakoid membrane and the four major complexes that it binds. The composition of the photosynthetic apparatus is continually changing in response to a range of environmental stimuli. In addition, many photosynthetic components have some of the highest turnover rates in Nature. Changes in composition and turnover of photosynthetic components require the degradation of existing and damaged polypeptides and the resynthesis and co-ordinated assembly of new polypeptides and their associated cofactors. This is achieved by several auxiliary functions, including proteolysis, protein targeting and the action of molecular chaperones. Some of the components involved in these functions, such as translocons, chaperones and proteases, have been identified but many of the auxiliary functions of photosynthesis remain uncharacterized. Among the proteases known to be associated with the thylakoids is the zinc metalloprotease FtsH, which might also act as a chaperone. Here we provide an overview of the thylakoid FtsH protease and discuss its role in the maintenance and assembly of the photosynthetic apparatus.

ATP-Dependent Proteases↗

Evolutionary relationships among photosynthetic prokaryotes (Heliobacterium chlorum, Chloroflexus aurantiacus, cyanobacteria, Chlorobium tepidum and proteobacteria): implications regarding the origin of photosynthesis.

The presence of shared conserved insertions or deletions in proteins (referred to as signature sequences) provides a powerful means to deduce the evolutionary relationships among prokaryotic organisms. This approach was used in the present work to deduce the branching orders of various eubacterial taxa consisting of photosynthetic organisms. For this purpose, portions of the Hsp60 and Hsp70 genes, covering known signature sequence regions, were PCR-amplified and sequenced from Heliobacterium chlorum, Chloroflexus aurantiacus and Chlorobium tepidum. This information was integrated with sequence data for several other proteins from numerous species to deduce the branching orders of different photosynthetic taxa. Based on signature sequences that are present in different proteins, it is possible to infer that the various eubacterial phyla evolved from a common ancestor in the following order: low G+C Gram-positive (H. chlorum) --> high G+C Gram-positive --> Deinococcus-Thermus --> green non-sulphur bacteria (Cf. aurantiacus ) --> cyanobacteria --> spirochaetes --> Chlamydia-Cytophaga-Aquifex-flavobacteria-green sulphur bacteria (Cb. tepidum) --> proteobacteria (alpha, delta and epsilon) and --> proteobacteria (beta and gamma). The members of the Heliobacteriaceae family that contain a Fe-S type of reaction centre (RC-1) and represent the sole photosynthetic phylum from the Gram-positive or monoderm group of prokaryotes are indicated to be the most ancestral of the photosynthetic lineages. Among the Gram-negative bacteria or diderm prokaryotes, green non-sulphur bacteria such as Cf. aurantiacus, which contains a pheophytin-quinone type of reaction centre (RC-2), are indicated to have evolved very early. Thus, the organisms containing either RC-1 or RC-2 existed before the evolution of cyanobacteria, which contain both these reaction centres to carry out oxygenic photosynthesis. The eubacterial divisions consisting of green sulphur bacteria and proteobacteria are indicated to have diverged after cyanobacteria. Some implications of these results concerning the origin of photosynthesis and the earliest prokaryotic fossils are discussed.

Amino Acid Sequence↗

In vitro activation and repression of photosynthesis gene transcription in Rhodobacter capsulatus.

It has been known for over half a century that anoxygenic photosynthetic bacteria maximally synthesize their photosystems in the absence of oxygen. During the last decade, it has become clear that this regulation is largely at the transcriptional level, with photosynthesis genes expressed only under anaerobic conditions. We describe here in vitro reconstitution of activation and repression of three photosynthesis promoters, bch (bacteriochlorophyll biosynthesis), puc (light-harvesting II apoproteins) and puf (reaction centre and light-harvesting I apoproteins) using purified transcription factors and RNA polymerase from Rhodobacter capsulatus. Previous genetic results have indicated that each of these three promoters is differentially regulated by three key regulators: CrtJ acting as a repressor of bch and puc and the two-component regulators RegA/RegB, which are activators of puc and puf. These regulators are distinct from those that mediate oxygen control in enteric bacteria. Our in vitro studies show that these purified regulators directly control the expression of the housekeeping RNA polymerase at these promoters. High-level basal expression of the bch promoter is shown to be repressed by CrtJ. The puc promoter is activated by the RegB-phosphorylated RegA protein and additionally repressed by CrtJ. At the puc promoter, CrtJ effectively competes for promoter binding with RegA, while at the bch promoter, repression appears to be by competition for the RNA polymerase binding site. In contrast to what has been suggested previously, the RegA-activated puf promoter is demonstrated as being recognized by the housekeeping RNA polymerase. We also discuss evidence that RegA approximately P activation of the puc and puf promoters involves recruitment of RNA polymerase by different modes of protein-protein interaction.

Bacterial Proteins↗

A moderate decrease of plastid aldolase activity inhibits photosynthesis, alters the levels of sugars and starch, and inhibits growth of potato plants.

Antisense expression of a full length cDNA encoding plastid aldolase led to decreased expression of aldolase at the transcript and protein level in several 'antisense' potato transformants. To quantify the inhibition, activity was compared in corresponding leaves down a plant and in plants of different ages. Aldolase activity was decreased by 32-43%, 56-71%, 79-83% and 91-97% in A-70, A-3, A-51 and A-2. Separation on a Q-Sepharose-FF column showed the decrease was due to inhibition of plastid aldolase. The transformants showed a small increase of Rubisco activity, a small decrease of phosphoribulokinase activity, and larger but subproportional decreases of sedoheptulose-1,7-biphosphatase and plastid fructose-1,6-bisphosphatase activity. Ambient photosynthesis was inhibited by 10%, 40%, 66% and 85% in A-70, A-3, A-51 and A-2. The transformants contained increased triose phosphates, and very low ribulose-1,5-bisphosphate and glycerate-3-phosphate. Chlorophyll fluorescence indicated that photosystem II was more reduced and thylakoid energization was increased. Starch synthesis was decreased by 16% and 36% in A-70 and A-3, whereas sucrose synthesis was less strongly inhibited. Plant growth was not significantly altered in A-70, was decreased by 41% in A-3, and was severely inhibited in plants with under 20% of wild-type aldolase activity. Although plastid aldolase catalyses a readily reversible reaction, possesses no known regulatory properties, and would appear irrelevant for the control of metabolism and growth, small changes in its activity have marked consequences for photosynthesis, carbon partitioning and growth.

Carbohydrate Metabolism↗

Rapid and proven production of transplastomic tobacco plants by restoration of pigmentation and photosynthesis.

Tobacco chloroplast transformation is typically achieved using dominant, selectable antibiotic resistance genes such as aadA, nptII and aphA-6. An improvement would be the combination of such a marker with a visual screening system for the early and conclusive detection of plastid transformants. As such, we investigated the use of three photosynthesis-deficient plastid mutants, DeltapetA, Deltaycf3 and DeltarpoA, for the development of a phenotypic selection system. Mutant plants were used as an alternative to the wild-type as source tissue for transformation, re-introducing deleted plastid sequences and using the aphA-6 gene as a selection marker. The reconstitution of the deleted genes in transformed regenerants resulted in shoots with a visually distinct phenotype comparable to the wild-type. This transformation/selection system overcomes the common problems associated with plastid transformation, e.g. the recovery of spontaneous mutants or nuclear insertions. In addition to the benefits offered by phenotypic selection, phenotype reconstitution leads to restoration of photosynthesis, which we assume drives reconstituted plants rapidly towards homoplasmy. As such, repeated cycles of regeneration in the presence of an antibiotic selection agent are no longer required.

Base Sequence↗

Direct and indirect climate change effects on photosynthesis and transpiration.

Climate change affects plants in many different ways. Increasing CO(2) concentration can increase photosynthetic rates. This is especially pronounced for C(3) plants, at high temperatures and under water-limited conditions. Increasing temperature also affects photosynthesis, but plants have a considerable ability to adapt to their growth conditions and can function even at extremely high temperatures, provided adequate water is available. Temperature optima differ between species and growth conditions, and are higher in elevated atmospheric CO(2). With increasing temperature, vapour pressure deficits of the air may increase, with a concomitant increase in the transpiration rate from plant canopies. However, if stomata close in response to increasing CO(2) concentration, or if there is a reduction in the diurnal temperature range, then transpiration rates may even decrease. Soil organic matter decomposition rates are likely to be stimulated by higher temperatures, so that nutrients can be more readily mineralised and made available to plants. This is likely to increase photosynthetic carbon gain in nutrient-limited systems. All the factors listed above interact strongly so that, for different combinations of increases in temperature and CO(2) concentration, and for systems in different climatic regions and primarily affected by water or nutrient limitations, photosynthesis must be expected to respond differently to the same climatic changes.

Carbon Dioxide↗

The effect of ultraviolet radiation on ultrastructure and photosynthesis in the red macroalgae Palmaria palmata and Odonthalia dentata from Arctic waters.

In radiation exposure experiments, the effects of mild artificial UV conditions (4.7 W m(-2) UV-A and 0.20 W m(-2) UV-B) plus PAR (25 - 30 micromol photons m(-2) s(-1)) on photosynthesis and ultrastructure of two red algal species from the Arctic have been investigated. While Palmaria palmata was collected from the upper sublittoral of the Kongsfjord (Spitsbergen, Norway), Odonthalia dentata represents a typical deepwater species at this high latitude. After 6 h and 24 h exposure to UV, chlorophyll fluorescence of photosystem II (PS II efficiency, F(v)/F(m)) was determined as an indicator for photosynthetic performance, and the relative electron transport rates in response to increasing photon fluence rates were recorded. In parallel, tissue samples were prepared for the transmission electron microscope (TEM). The presented data clearly demonstrate a significant influence of experimental UV on photosynthetic performance. Photochemical efficiency of PS II of both red algal species decreased to about one third of the initial value under UV. While the PI (photosynthesis-irradiance) curve parameter alpha (positive slope at limiting photon fluence rates) strongly decreased in both plants, the I(k) values (initial value of light-saturated photosynthetic rate) increased 3 - 5-fold. Palmaria palmata does not appear to become photoinhibited under these conditions, but O. dentata showed strong photoinhibition. The TEM results demonstrated that the photosynthetic apparatus was severely influenced by UV in both species, because thylakoid membranes appeared wrinkled, lumen dilatations occurred, and the outer membranes were altered. Moreover, mitochondria were damaged, and numerous plasma vesicles were observed. In conclusion, both red algal species are negatively affected by UV on the physiological and ultrastructural level. However, the differences in photoinhibitory responses correlate well with the vertical depth zonation of P. palmata and O. dentata in the Arctic Kongsfjord.

Arctic Regions↗

Stomatal conductance is a key parameter to assess limitations to photosynthesis and growth potential in barley genotypes.

Fourteen genotypes of barley were compared for response to salinity by monitoring the parameters gas exchange and chlorophyll fluorescence. We present relationships between stomatal conductance (gs) gas exchange chlorophyll fluorescence parameters and aboveground dry matter (AGDM). We found that genetic variability provided a continuum of data for gs across control and saline conditions. We used this continuum of gs values to test the overall relationships between gs and net photosynthesis (A), leaf internal CO2 concentration (Ci), actual quantum yield of PSII electron transport (PhiPSII), relative electron yield over net CO2 assimilation rate (ETR/A), and AGDM. The relationship between gs and A was highly significant (P < 0.0001) for both control and saline treatments, while correlations between gs and Ci, and Ci and A were significant only under control conditions. Unexpectedly, we found positive correlations between gs and PhiPSII (P < 0.0001) for both conditions. A comparison between relationships of gs and A, and gs and PhiPSII seemed to indicate a possible acclimation to salinity at the chloroplastic level. Finally, the relationships between gs and ETR/A were exceptionally strong for both growing conditions (P < 0.0001) indicating that, as gs values were negatively affected in barley by genetics and salinity as main or interactive effects, there was a progressive increase in photorespiration in barley. Overall, we found that stomatal conductance was a key parameter in the study of barley responses to limiting situations for photosynthesis. We also found a strong relationship between AGDM and gs regardless of growing conditions and genotypes. For breeding evaluations to select barley genotypes for salinity tolerance, it may be possible to replace all measurements of gas exchange and chlorophyll fluorescence by the simple use of a porometer.

Adaptation, Physiological↗

The Paleoproterozoic snowball Earth: a climate disaster triggered by the evolution of oxygenic photosynthesis.

Although biomarker, trace element, and isotopic evidence have been used to claim that oxygenic photosynthesis evolved by 2.8 giga-annum before present (Ga) and perhaps as early as 3.7 Ga, a skeptical examination raises considerable doubt about the presence of oxygen producers at these times. Geological features suggestive of oxygen, such as red beds, lateritic paleosols, and the return of sedimentary sulfate deposits after a approximately 900-million year hiatus, occur shortly before the approximately 2.3-2.2 Ga Makganyene "snowball Earth" (global glaciation). The massive deposition of Mn, which has a high redox potential, practically requires the presence of environmental oxygen after the snowball. New age constraints from the Transvaal Supergroup of South Africa suggest that all three glaciations in the Huronian Supergroup of Canada predate the Snowball event. A simple cyanobacterial growth model incorporating the range of C, Fe, and P fluxes expected during a partial glaciation in an anoxic world with high-Fe oceans indicates that oxygenic photosynthesis could have destroyed a methane greenhouse and triggered a snowball event on time-scales as short as 1 million years. As the geological evidence requiring oxygen does not appear during the Pongola glaciation at 2.9 Ga or during the Huronian glaciations, we argue that oxygenic cyanobacteria evolved and radiated shortly before the Makganyene snowball.

Biological Evolution↗

Functional differences between galactolipids and glucolipids revealed in photosynthesis of higher plants.

Galactolipids represent the most abundant lipid class in thylakoid membranes, where oxygenic photosynthesis is performed. The identification of galactolipids at specific sites within photosynthetic complexes by x-ray crystallography implies specific roles for galactolipids during photosynthetic electron transport. The preference for galactose and not for the more abundant sugar glucose in thylakoid lipids and their specific roles in photosynthesis are not understood. Introduction of a bacterial glucosyltransferase from Chloroflexus aurantiacus into the galactolipid-deficient dgd1 mutant of Arabidopsis thaliana resulted in the accumulation of a glucose-containing lipid in the thylakoids. At the same time, the growth defect of the dgd1 mutant was complemented. However, the degree of trimerization of light-harvesting complex II and the photosynthetic quantum yield of transformed dgd1 plants were only partially restored. These results indicate that specific interactions of the galactolipid head group with photosynthetic protein complexes might explain the preference for galactose in thylakoid lipids of higher plants. Therefore, galactose in thylakoid lipids can be exchanged with glucose without severe effects on growth, but the presence of galactose is crucial to maintain maximal photosynthetic efficiency.

Arabidopsis↗

The origin of atmospheric oxygen on Earth: the innovation of oxygenic photosynthesis.

The evolution of O(2)-producing cyanobacteria that use water as terminal reductant transformed Earth's atmosphere to one suitable for the evolution of aerobic metabolism and complex life. The innovation of water oxidation freed photosynthesis to invade new environments and visibly changed the face of the Earth. We offer a new hypothesis for how this process evolved, which identifies two critical roles for carbon dioxide in the Archean period. First, we present a thermodynamic analysis showing that bicarbonate (formed by dissolution of CO(2)) is a more efficient alternative substrate than water for O(2) production by oxygenic phototrophs. This analysis clarifies the origin of the long debated "bicarbonate effect" on photosynthetic O(2) production. We propose that bicarbonate was the thermodynamically preferred reductant before water in the evolution of oxygenic photosynthesis. Second, we have examined the speciation of manganese(II) and bicarbonate in water, and find that they form Mn-bicarbonate clusters as the major species under conditions that model the chemistry of the Archean sea. These clusters have been found to be highly efficient precursors for the assembly of the tetramanganese-oxide core of the water-oxidizing enzyme during biogenesis. We show that these clusters can be oxidized at electrochemical potentials that are accessible to anoxygenic phototrophs and thus the most likely building blocks for assembly of the first O(2) evolving photoreaction center, most likely originating from green nonsulfur bacteria before the evolution of cyanobacteria.

Atmosphere↗

Primary charge separation in bacterial photosynthesis: oxidized chlorophylls and reduced pheophytin.

Bacteriopheophytin, the magnesium-free base of bacteriochlorophyll, undergoes reversible one-electron reduction in organic solvents to yield an anionic free radical with characteristic optical and electron spin resonance spectra. The reduction potential of bacteriopheophytin, E1/2 approximately --0.55 V against a normal hydrogen electrode, compared to E1/2 approximately --0.85 V for bacteriochlorophyll, renders it a likely electron acceptor in the primary charge separation of photosynthesis. Comparison of these data with picosecond optical changes recently observed upon pulsed laser excitation of bacterial reaction centers leads us to propose that bacteriopheophytin is indeed a transient electron acceptor and that the primary charge separation of bacterial photosynthesis occurs between the bacteriochlorophyll complex P870 and bacteriopheophytin to yield the radicals of the oxidized chlorophyll dimer cation and reduced pheophytin anion.

Bacteriochlorophylls↗

Eubacteria, halobacteria, and the origin of photosynthesis: the photocytes.

The halobacteria and the photosynthetic members of the eubacteria have previously been classified in two separate urkingdoms--the archaebacteria and the eubacteria, respectively. They were thought to be no more closely related to each other than they each were to the eukaryotes. In accord with this earlier classification, photosynthesis was thought to have originated twice by independent events--once within the eubacteria and once within the archaebacteria. In this paper, however, using three-dimensional ribosome structure as a probe of evolutionary divergences, we show that the eubacteria and the halobacteria are more closely related to each other than they are to any other known organisms. The simplest interpretation of our data is that all extant photosynthetic cells are descended from a single common ancestor that possessed a primeval photosynthetic mechanism. Numerous data on the occurrence of related biochemical processes in halobacteria and eubacteria support this theory. Essential components of the photosynthetic apparatus, such as carotenoids, are present in both halobacteria and in eubacteria, including the nonphotosynthetic eubacteria, suggesting that photosynthesis could be a primitive property of both groups. Our data indicate that together the eubacteria and the halobacteria form a monophyletic group for which we propose the name "photocytes." If other techniques of phylogenetic analysis confirm this evolutionary tree, we propose that the photocytes be given urkingdom status.

Biological Evolution↗

Restoration of phototrophic growth in a mutant of Chlamydomonas reinhardtii in which the chloroplast atpB gene of the ATP synthase has a deletion: an example of mitochondria-dependent photosynthesis.

The Chlamydomonas reinhardtii mutant FUD50 has a deletion in the atpB gene of the chloroplast ATP synthase [Woessner, J. P., Masson, A., Harris, E. H., Bennoun, P., Gillham, N. W., and Boynton, J. E. (1984) Plant Mol. Biol. 3, 177-190]. We have isolated a suppressed strain (FUD50su) that can grow under phototrophic conditions, although it still showed no synthesis of the beta subunit of coupling factor 1. Thylakoid membranes of the FUD50su strain were similar to those of the original FUD50 strain, in that they both lacked all the subunits making up the chloroplast ATP synthase complex. We show that photosynthesis in FUD50su is sensitive to inhibitors such as antimycin, specific for mitochondrial electron transport. This observation indicates that photosynthesis in the FUD50su strain is achieved through an unusual interaction between mitochondria and chloroplast. Exportation of light-induced reduced compounds from the chloroplast to the mitochondria elicits ATP formation in the latter, and ATP is subsequently imported to the chloroplast. The activation of such an ATP shuttle coupled to an NADPH shuttle would thus provide the reducing power and the free energy needed for carbon assimilation in a chloroplast that lacks chloroplast ATP synthase.

Chlamydomonas↗

DNA binding characteristics of RegA. A constitutively active anaerobic activator of photosynthesis gene expression in Rhodobacter capsulatus.

In the purple non-sulfur bacterium Rhodobacter capsulatus, RegA and RegB comprise a two-component regulatory system that is required for maximal anaerobic transcription of key photosynthesis genes. RegB is a sensor kinase that uses ATP to phosphorylate its cognate response regulator, RegA. The mechanism under which RegA approximately P influences transcription of target genes has been unclear given that past attempts to demonstrate DNA binding activity by isolated RegA have failed. This led to a model invoking a role for RegA approximately P as an intermediate in a more complex multicomponent phosphoryl transfer cascade. In the present study, we describe the isolation of a mutant version of RegA (RegA*) which promotes high level expression of photosynthesis genes independent of RegB. DNase I footprint analyses show that purified RegA* binds to the promoters of the puf and puc operons at locations that are consistent with RegA functioning as a transcriptional activator for these operons. We conclude that RegA functions, like most members of the response regulator family, as a DNA-binding protein that directly affects the expression of its target genes.

Amino Acid Sequence↗

A novel mechanism for the regulation of photosynthesis gene expression by the TspO outer membrane protein of Rhodobacter sphaeroides 2.4.1.

A bacterial homolog of the mammalian mitochondrial benzodiazepine receptor, the tryptophan-rich sensory protein (TspO) has been previously demonstrated to negatively affect the transcriptional expression of several photosynthesis genes of Rhodobacter sphaeroides. To identify components of the signal transduction pathway from the outer membrane-localized TspO to the DNA-active transcription factor(s), we examined the involvement of TspO in the regulation of tetrapyrrole metabolism in R. sphaeroides. By analyzing the tetrapyrrole pigments accumulated by resting cell suspensions of R. sphaeroides, we demonstrated that TspO negatively regulates the activity of coproporphyrinogen III oxidase in this bacterium. hemN, encoding one of the isoenzymes of coproporphyrinogen III oxidase of R. sphaeroides, provided in trans to the wild type strain, produced a TSPO1 mutant phenotype by abolishing the negative effect of TspO on the transcription of the photosynthesis genes, crtI and puc. It is proposed that TspO, by regulating the exit of certain tetrapyrrole intermediates of the heme/bacteriochlorophyll biosynthetic pathways in R. sphaeroides in response to the availability of molecular oxygen, causes the accumulation of a biosynthetic intermediate that serves as a corepressor for both specific pigment gene transcription and the puc operon. The relationship between the bacterial TspO and the mitochondrial peripheral benzodiazepine receptor is discussed.

Animals↗