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Some effects of light on the interconversion of metabolites in green

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... A tendency for the formation ofmalate to be more pronounced in the light was shown also in a series of experiments by Graham & Walker (1962). They fed 14C-labelled acids to mung-bean leaves and found that more label appeared in malate in the light than in the dark. ...
... (In our experiments the total amount of aspartic acid fell slightly during the period of illumination, and an increase in organic nitrogen consisted chiefly ofa conversion of glutamic acid into glutamine.) Graham & Walker (1962) suggested that photosynthesis kept a greater proportion of NAD in the reduced state and thus with the participation of malate dehydrogenase diverted more of the label reaching oxaloacetate into the malate pool. In the dark, metabolism would shift to a more oxidative condition and formation of oxaloacetate (aspartic acid) and citrate would be favoured. ...
Article
1. Mustard plants have been grown under conditions in which the length of artificial day could be controlled. 2. Leaf samples were analysed for malic acid and citric acid, and for a number of inorganic anions and cations. A simple method is described by which sap was obtained from 0.5g. samples of leaves. 3. In days of 16hr. or more, malic acid was accumulated; the chief cation accumulated was calcium. 4. When the day-length was reduced the malic acid content decreased considerably but the calcium content remained the same. There was little change in the pH value of the sap, the balance of anions and cations having been maintained mainly by increases in citrate and nitrate contents. Analyses of the whole leaf still showed some deficiency in anion after sodium, potassium, calcium, magnesium, nitrate, sulphate, inorganic phosphate, chloride, malate and citrate had been accounted for. 5. Analyses at shorter intervals revealed a large diurnal variation in malic acid content, which increased during the first 5-6hr. of the light period, and fell during darkness. 6. The significance of these findings is discussed, and it is suggested that malic acid accumulation is a by-product of photosynthesis, calcium being taken up irreversibly to maintain anion/cation balance, and hence creating a continuing need for anions to balance it.
... At the time, such results appeared to be in contradiction with fatty acids (FA) being 14 C-labelled upon 14 CO 2 feeding (in illuminated leaves and chloroplasts; Stumpf & James, 1962, simply because FA production requires glycolytic degradation of glucose to pyruvate. Subsequent labelling with 14 C metabolites (including citrate or fumarate) suggested that the flux through the Krebs cycle decreased transiently upon the dark-to-light transition but operated at a similar rate in the light and in the dark (Graham & Walker, 1962;Chapman & Graham, 1974a,b). A typical difference between light and dark was that oxaloacetate appeared to be converted to malate in the light while it yielded aspartate in the dark. ...
... A typical difference between light and dark was that oxaloacetate appeared to be converted to malate in the light while it yielded aspartate in the dark. This was interpreted as being the result of excess reductive power slowing down the 'left branch' of the Krebs cycle (Graham & Walker, 1962;Chapman & Graham, 1974a). Experiments monitoring 14 CO 2 evolution in CO 2 -free air further suggested that respiratory CO 2 efflux was inhibited by c. 75% in the light (Mangat et al., 1974). ...
Article
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Contents I. II. III. IV. V. VI. VII. VIII. References SUMMARY: It has been 75 yr since leaf respiratory metabolism in the light (day respiration) was identified as a low-flux metabolic pathway that accompanies photosynthesis. In principle, it provides carbon backbones for nitrogen assimilation and evolves CO2 and thus impacts on plant carbon and nitrogen balances. However, for a long time, uncertainties have remained as to whether techniques used to measure day respiratory efflux were valid and whether day respiration responded to environmental gaseous conditions. In the past few years, significant advances have been made using carbon isotopes, 'omics' analyses and surveys of respiration rates in mesocosms or ecosystems. There is substantial evidence that day respiration should be viewed as a highly dynamic metabolic pathway that interacts with photosynthesis and photorespiration and responds to atmospheric CO2 mole fraction. The view of leaf day respiration as a constant and/or negligible parameter of net carbon exchange is now outdated and it should now be regarded as a central actor of plant carbon-use efficiency.
... Oh-hama and Miyachi (1960) have shown that illumination has marked effects on their levels in Ohlorella, and very recently changes in levels of these compounds during lightdark transitions in Beta, Spinacia, and Elodea have been reported by Heber and Santarius (1965). Further, Graham and Walker (1962) showed that illumination of green leaves of mung bean resulted in considerable changes in the levels of intermediates of the Krebs cycle and related compounds, particularly malate and aspartate, and it was suggested that this was caused by changes in the ratio of oxidized to reduced nicotinamide adenine nucleotides. In the work .now ...
... Our data provide support for the postulation of Graham and Walker (1962) that the reciprocal changes in the malate/aspartate ratio occurring between darkness and illumination are explainable in terms of a lower ratio of NAD/NADH2 in the light than in the.dark. ...
Article
Changes in levels of nicotinamide adenine nucleotides were measured during a short (30 min) period of illumination following a dark period. Two phases in the time course were found. In the first phase, during the first minute of illumination, a rapid decline in oxidized nicotinamide adenine dinucleotide occurred which represented a net loss of nicotinamide adenine nucleotide. In the subsequent, second phase during illumination, a slower decline in oxidized nicotinamide adenine dinucleotide was found which was coincident with increases in nicotinamide adenine dinucleotide phosphate(s). The changes in the reduced nicotinamide adenine nucleo· tides were relatively small during illumination.
... The PEPc reaction in leaves of C 3 -plants is activated under illumination (Duff & Chollet, 1995 ) to replenish the carbon skeletons used for biosyntheses. The concentration of oxaloacetate (from PEPc) and NADH (from photorespiration ) rise and shift the mtMDH reaction towards malate production (Graham & Walker, 1962). MtME in turn is down-regulated (Hill & Bryce, 1992; Igamberdiev et al., 2001) and thus malate levels in leaves are highest at the end of the light period. ...
... discussion in Barbour et al., 2011). After 'deactivation' of mtME in the course of darkening, the anapleurotic TCA activity can lead to a decreasing malate and an increasing citrate concentration in autotrophic cells (Graham & Walker, 1962). The maximum CO 2 production of the reconstituted TCA cycle per conversion cycle (without mtME) is then three CO 2 molecules (one each from mtPDH, mtIDH and mt2-OGDH) out of seven carbon atoms (four malate-and three pyruvate-carbon;Fig. ...
... The rise and fall of 14 C-incorporation in aspartic acid was in the reverse direction with the 14 C-incorporation that occurred in malic acid under the similar conditions. Graham and Walker (1962) reported that with Mung bean, strong light led to ...
Article
The moss, Dicranum scoparium, conditioned and maintained at temperatures ranging from 0° to 10°C, was subjected to acetate-2-14C incubation at 5° and 22°C in the presence or absence of added illumination. Patterns of 14C-incorporation into various non-protein fractions (including lipid-fatty acids) respired CO2, and alcoholic solubles were examined. The 14C-labeled metabolic products in amino acid-, organic acid-, and sugar-pools were separated and identified with column and thinlayer chromatographic techniques. Radioactivity increased in the sugar-pool under illumination regardless of incubation temperatures. The radioactivity in glucose remained unchanged regardless of illumination, but the formation of sucrose appeared to be light regulated. The combined regime of added illumination and raised temperature brought about an increase in activity for organic acid and a decrease in amino acid-pools although a sharp rise of 14C-glutamine was occurring in the latter pool. High light gave rise to more 14C-amino acids than those with a low light, regardless of incubation temperatures. Total level of activity in lipid-fatty acid pool remained unchanged during changes of illumination and incubation temperature, while its components, Lx and L2, fluctuated.
... The extraction procedure was a modification of that used by Graham and Walker (1962). Each sample of O. vulgari8 was extracted for 3 min in boiling 80% (v/v) ethanol and allowed to stand for 5 min. ...
Article
When O. vulgaris is treated in complete darkness with diquat at concentrations which are toxic in the light, there is a rapid and large stimulation of oxygen uptake with no significant alteration in the R,Q. If the O. vulgaris has been darkened for some hours before the addition of diquat there is a two- to threefold stimulation of the rate of respiration, which subsequently remains constant or falls only slowly for up to 500 min. With pre-illuminated or glucose-treated O. vulgaris, diquat causes a much greater stimulation of dark respiration, rapidly declining to near the control rate. Tracer studies with 14C02 and [14C]glucose show that diquat when added in darkness to O. vulgaris accelerates the breakdown of starch and the loss of 14C from most intermediate substances of carbohydrate metabolism. The exception to this is the rapid accumulation of 14C counts in citric acid. These results are discussed and a mechanism for the action of diquat in darkness is proposed.
... This result is in accordance with data reported for tobacco (5) and sugar beet (17). The rapid decrease in the daytime might be attributable to increased conversion of aspartate into other amino acids and malate (18). The increase in the night might be attributed to dark C0 2 fixation and transamination of oxalacetate with glutamate acting as the primary amino donor. ...
Article
Greenhouse-grown soybean (Glycine max L.) plants were subjected to water stress and diurnal changes in the contents of free amino acids were determined. In non-stressed plants with full water supply, the major parts of the free amino acids showed a similar diurnal variation, and the maximum contents were obtained between 11 a.m. and 2 p.m. except for aspartate. The maximum content of aspartate was obtained during the night. Glycine, serine and alanine, in particular, showed a light-sensitive response.In water-stressed plants, the water potential of the leaves fell from -12.8 bars at 7 a.m. to -22.2 bars at 7 p.m. and returned to -19.0 bars after sunset. When the leaf water potential fell, the total protein decreased with increase in the total free amino acids.Water stress induced a remarkable accumulation of free amino acids: histidine, valine, isoleucine, leucine, tyrosine, phenylalanine, and especially proline and asparagine. The maximum contents of proline and asparagine were 31 and 24% of the total free amino acid nitrogen, respectively. The contents of lysine, arginine, aspartate, glutamate, glutamine, glycine and alanine did not show a large increase. These results indicate a difference in turnover rate of amino acids after liberation by proteolysis.The ammonia content of the water-stressed plants was consistently low during 24 hr.
Article
Tobacco plants (Nicotiana tabacum L.) were given 5 min of red (R) or far-red (FR) radiation at the end of each day, following full intensity long (16-hr) and short (8-hr) photoperiods. Contents of free sugars, organic acids and amino acids in the plants were examined along with photo-induced differences in morphological development. Plants under long photoperiods grew more than those under short photoperiods. Within each photoperiod, plants irradiated with R developed shorter stems and darker green leaves than did those irradiated with FR. Long photoperiods resulted in higher concentrations of free sugars and lower concentrations of organic acids than did short photoperiods. Within each photoperiod, plants receiving terminal FR radiation had higher concentrations of free sugars and organic acids than did those receiving terminal R radiation. Plants grown under short photoperiods had a higher amino acid concentration than did those under long photoperiods. Concentration of amino acids in plants grown under long photoperiods was not affected by terminal R or FR. However, under short photoperiods plants irradiated with R had higher amino acid concentrations than did those irradiated with FR.
Article
In this report, properties of isolated mitochondria from pea leaves have been studied in view to their function in photosynthesis metabolism of a leaf cell. (1) The rates of respiration with various substrates and with a combination of these substrates have been measured with isolated mitochondria. The highest respiration rate was found with NADH, followed by NADPH, malate and glycine. For the oxidation of NADH, NADPH, malate, glycine and 2-oxoglutarate the apparent Km values were determined. The oxidation of malate and glycine occurred independently of each other as long as electron transport was not limiting. (2) The maximal capacity of mitochondrial ATP synthesis in a leaf was estimated as about 25% of the rate of noncyclic photophosphorylation at maximal rate of photosynthesis. (3) From measurements of NADH/NAD ratios in isolated mitochondria and from previous determinations of the NADH/NAD ratio in the cytosol of spinach leaves it is discussed that in a leaf cell the NADH/NAD ratio in the mitochondrial matrix is higher than in the cytosol. (4) A comparison of the apparent Km values obtained for the oxidation of external NADH and NADPH with the corresponding concentrations found in the cytosol of spinach leaves suggests that in a leaf cell NADPH is oxidized by mitochondria at a much higher rate than NADH. (5) From the measurement of mitochondrial respiration with glycine and malate as substrates in the presence of a defined malate / oxaloacetate ratio the function of a malate-oxaloacetate shuttle is demonstrated. It is furthermore shown that a malate-aspartate shuttle does not play any significant role in redox transfer under physiological conditions.
Article
Photosynthesis and respiration in an illuminated plant cell are not only interdependent but also mutually beneficial. Respiratory rates increase after hours of illumination due to carbohydrate (substrate) accumulation. Besides such long-term effects, photosynthesis and respiration interact even during short illumination periods of a few minutes. The rate of respiration in isolated leaf protoplasts increases severalfold after 10–15 min of illumination. Such light-enhanced dark respiration (LEDR) has been demonstrated in protoplasts as well as in leaves. The stimulation of LEDR by bicarbonate and its sensitivity to inhibitors of photosynthesis (DCMU) or the Calvin cycle (d,l-glyceraldehyde point out the importance of photosynthetic carbon metabolism for respiration. From metabolite analyses of protoplasts, the majority of LEDR is due to mitochondrial oxidation of malate produced by chloroplasts. Simultaneous measurements of photosynthesis and respiration, using mass spectrometry, demonstrate that mitochondrial TCA cycle-based CO2 evolution is inhibited by illumination while O2 uptake is either unaffected or stimulated. The marked sensitivity of photosynthesis in leaves or protoplasts to classic mitochondrial inhibitors such as oligomycin, sodium azide or antimycin A implies that mitochondrial metabolism is essential for photosynthesis. Respiration not only benefits photosynthesis but also protects illuminated leaf protoplasts against photoinhibition. Oxidative electron transport and phosphorylation play a much more important role than the reactions of glycolysis and the TCA cycle in this beneficial interaction. The metabolite shuttles involving PGA-DHAP and/or OAA-malate across the chloroplast and mitochondrial membranes could form the biochemical basis of the interaction between photosynthesis and respiration. Alternatively, cytosolic NAD(P)H, derived from photosynthetic products, can be directly acted upon by the mitochondrial external NAD(P)H dehydrogenase and oxidised through the mitochondrial electron transport system. Mitochondrial oxidation of NAD(P)H (even if indirect) helps to prevent the over-reduction of the cytosol and, consequently, the chloroplast in illuminated leaf cells. Besides the direct interaction with chloroplasts, mitochondria can supply reducing equivalents through malate to peroxisomes during photorespiration and provide citrate as the precursor of oxoglutarate, necessary for glutamine and glutamate formation. These two phenomena further complement the strong interdependence of photosynthesis and mitochondrial metabolism.
Article
The effect of light on oxygen uptake and production by algae has been studied. Illumination was found to influence oxygen uptake by two mechanisms. Oxygen uptake was found to be inhibited at low light intensities and accelerated at medium to high intensities. The inhibition of uptake was mainly sensitized by chlorophyll a. The effects of starvation, glucose supplementation, and DCMU inhibition on the oxygen uptake rates in the light are described.
Article
1. The relative efficiencies of nicotinate, quinolinate and nicotinamide as precursors of NAD(+) were measured in the first leaf of barley seedlings. 2. In small amounts, both [(14)C]nicotinate and [(14)C]quinolinate were quickly and efficiently incorporated into NAD(+) and some evidence is presented suggesting that NAD(+) is formed from each via nicotinic acid mononucleotide and deamido-NAD. 3. [(14)C]Nicotinamide served equally well as a precursor of NAD(+) and although significant amounts of [(14)C]NMN were detected, most of the [(14)C]NAD(+) was derived from nicotinate intermediates formed by deamination of [(14)C]nicotinamide. 4. Radioactive NMN was also a product of the metabolism of [(14)C]nicotinate and [(14)C]quinolinate but most probably it arose from the breakdown of [(14)C]NAD(+). 5. In barley leaves where the concentration of NAD(+) is markedly increased by infection with Erysiphe graminis, the pathways of NAD(+) biosynthesis did not appear to be altered after infection. A comparison of the rates of [(14)C]NAD(+) formation in infected and non-infected leaves indicated that the increase in NAD(+) content was not due to an increased rate of synthesis.
Article
The relationship between the ATP/ADP ratios inside and outside the mitochondria was studied using silicon-layer centrifugation. As predicted from the properties of the adenine nucleotide translocation, the ATP/ADP ratio in the extramitochondrial space was found to be higher than in the mitochondria when in the state of respiratory control. Taking into account the content of inorganic phosphate, the phosphorylation potential in the mitochondria is calculated to be 2.3 kcal lower than in the medium.This difference between the internal and external ATP/ADP ratios is abolished if uncoupler is added, When there is also no Mg2+ present in the medium, the internal ATP/ADP ratio may be even higher than the external ratio, provided that there was oligomycin added in order to inhibit ATP hydrolysis.It is shown that the ADP is transported against a gradient from the extramitochondrial space into the mitochondria. The discussion of a model suggests that the physiological role of the energy-requiring transport of ADP into the mitochondria is to maintain a constant level of the ATP in the cytosol.
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The review emphasizes the essentiality of mitochondrial oxidative metabolism for photosynthetic carbon assimilation. Photosynthetic activity in chloroplasts and oxidative metabolism in mitochondria interact with each other and stimulate their activities. During light, the partially modified TCA cycle supplies oxoglutarate to cytosol and chloroplasts. The marked stimulation of O2 uptake after few minutes of photosynthetic activity, termed as light enhanced dark respiration (LEDR), is now a well-known phenomenon. Both the cytochrome and alternative pathways of mitochondrial electron transport are important in such interactions. The function of chloroplast is optimized by the complementary nature of mitochondrial metabolism in multiple ways: facilitation of export of excess reduced equivalents from chloroplasts, shortening of photosynthetic induction, maintenance of photorespiratory activity, and supply of ATP for sucrose biosynthesis as well as other cytosolic needs. Further, the mitochondrial oxidative electron transport and phosphorylation also protects chloroplasts against photoinhibition. Besides mitochondrial respiration, reducing equivalents (and ATP) are used for other metabolic phenomena, such as sulfur or nitrogen metabolism and photorespiration. These reactions often involve peroxisomes and cytosol. The beneficial interaction between chloroplasts and mitochondria therefore extends invariably to also peroxisomes and cytosol. While the interorganelle exchange of metabolites is the known basis of such interaction, further experiments are warranted to identify other biochemical signals between them. The uses of techniques such as on-line mass spectrometric measurement, novel mutants/transgenics, and variability in metabolism by growth conditions hold a high promise to help the plant biologist to understand this
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1.1. After illumination of leaves in the presence of 14CO2 for various times and subsequent freeze drying, chloroplasts were isolated using a nonaqueous procedure. The time-course of the distribution of a number of compounds between chloroplasts and the remainder of the cell was calculated from the 14C-incorporation into the fractions obtained.2.2. Labelled ribulose diphosphate, sedoheptulose diphosphate and sedoheptulose monophosphate occurred, at least during the first minutes of photosynthesis, solely in the chloroplasts. At the beginning of photosynthesis phosphoglyceric acid, fructose diphosphate, fructose monophosphate and glucose monophosphate appeared first in the chloroplasts, but were found later also in the non-chloroplastic part of the cell. The major part of glucose diphosphate, uridine diphosphoglucose, sucrose, malic acid and citric acid was always located in the non-chloroplastic part of the cell.3.3. From the results it is concluded that the photosynthetic carbon cycle operates exclusively in the chloroplasts. Sugar phosphates, which are not needed in the cyclic regeneration of the CO2-acceptor, are directly translocated into the cytoplasm. The synthesis of uridine diphosphoglucose takes place mainly in the cytoplasm. Glucose diphosphate and possibly also sucrose seem to be formed in the cytoplasm of the leaf cell.
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Metabolism of glutamic acid, γ-aminobutyric acid, and proline in tobacco leaves during the day and night has been studied by using isotopic labeled compounds. Biosynthesis of proline from glutamate was very active in the day but slow at night; this indicates that light has an important role in the synthesis. γ-Aminobutyrate was very readily transformed into organic acids, and most of the activity was found in malate, succinate, and citrate. Accordingly, in tobacco leaves γ-aminobutyrate seems to be oxidized to succinate via succinic semialdehyde and to enter the tricarboxylic acid cycle. Oxidation of proline to glutamate took place very slowly in tobacco leaves both in the day and night without any appreciable difference. Reactions of proline in the stem and roots were similar to those in leaves. Accumulation of the activity of glutamate and γ-aminobutyrate in aspartate and citrate was higher at night than in the day.
Article
Carbon dioxide fixation by leaf pieces from 12 different chloroplast mutants and wild type barley has been analysed. In the light leaf pieces from wild type seedlings fixed14CO2 at a rate of approximately 80 μmoles per gram fresh weight per hour, or 40 μmoles per mg chlorophyll per hour. Fixation of14CO2 in darkness occurred at one to four per cent of the rate in light. Five of the mutants investigated (vir-zb 63, vir-l27, vir-zd69, vir-c12 andxan-m 3) were completely blocked in light-dependent CO2 fixation. The mutantsvir-m 29, vir-u46, xan-d49, xan-c47, vir-t45, xan-t50 andvir-k 23 all yielded reduced photosynthetic rates on a gram fresh weight basis, but, with the exception ofvir-k 23, their photosynthetic rates on a chlorophyll basis differed by less than a factor of two from that of the wild type. On a chlorophyll basisvir-k 23 showed an enhanced rate of14CO2 fixation in comparison with the wild type. Barley mutants lacking light-dependent14CO2 incorporation fixed in darkness or in light similar small amounts of14CO2 into malate, aspartate, glutamate and citrate as did the wild type in darkness. Photosynthetic products in the mutants capable of light-dependent CO2 fixation were qualitatively the same as in the wild type, and the patterns of distribution of tracer carbon gave no evidence of any defects in reactions following the fixation of CO2. The gross correlation between chlorophyll contents and photosynthetic rates on a chlorophyll basis among these mutants with the exception ofvir-k 23 suggests that the reduced absorption of light by the lower amounts of chlorophyll in these mutants is the major or only factor responsible for their reduced photosynthetic rates on a gram fresh weight of leaf basis. The suggestion is made thatvir-k 23, which is characterized by a far higher photosynthetic rate in bright light than the wild type on a chlorophyll basis, contains several-fold lower amounts of light-harvesting chlorophyll per photosynthetic unit than wild type, whereas the other mutants that are capable of photosynthesis have photosynthetic units of the same size as the wild type but fewer of them.
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High concentrations of carbon dioxide inhibit the greening of etiolated plants. In the presence of 20% oxygen, concentrations of carbon dioxide of 10% and above inhibited the production of chlorophyll in etiolated leaves of barley, wheat, and dwarf French bean. On return to air, recovery from this inhibition took place rapidly. High concentrations of carbon dioxide were also inhibitory when illumination was discontinuous (2-msecond flash separated by 3-minute dark period) during which photosynthetic activity was adjudged to be negligible. The inhibition was alleviated by feeding with delta-amino levulinic aid, implying that the site of inhibition was early in the sequence of chlorophyll synthesis. 15 references, 3 figures, 1 table.
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Incorporation of C¹⁴Oâ during photosynthesis has been studied in healthy and rust-affected plants of bean, wheat, and safflower. Bean leaves carrying relatively light infection intensities showed consistent stimulation durin the flecking period when symptoms were first apparent. Subsequently, rate of C¹⁴Oâ incorporation declined to one-third or one-half the rates of normal leaves. Heavily infected primary bean leaves did not show stimulation at any time, but did exhibit an abrupt decline in C¹⁴Oâ fixation once sporulation was apparent. Wheat leaves infected by the stem rust fungus also showed marked inhibition during fungus sporulation, but there was no positive evidence for stimulated rates at any level of infection. Marked stimulation was found in young trifoliate leaves during periods when infected unifoliate leaves were inhibited. The extent of stimulation appeared to be related to the infection intensity of inoculated primary leaves. The data suggest that inhibition of photosynthesis in infected tissue can be compensated at least partially by stimulation in organs at a distance from the infected leaf. 25 references, 4 figures, 5 tables.
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Intact chloroplasts isolated from leaves of Vicia faba L. var. the Sutton show a decline in the endogenous level of alpha-ketoglutarate upon illumination. alpha-Ketoglutarate supplied to the chloroplasts is similarly utilized in this light-dependent reaction, and its consumption is paralleled by a concomitant increase in the level of glutamate. There is no photostimulation of glutamate synthesis in chloroplasts broken by osmotic shock, but it can be somewhat restored by addition of ferredoxin and NADP. These results suggest that in the isolated chloroplast the synthesis of glutamate from alpha-ketoglutarate is regulated by the availability of reduced pyridine nucleotide generated by photosynthetic electron transport. This conclusion is supported by the finding of an apparent competition between the photoreduction of phosphoglycerate to triose phosphate and the photoutilization of alpha-ketoglutarate.
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In leaves, the anaerobic accumulation of alanine was accompanied by a loss of aspartate, and these changes preceded gamma-aminobutyrate accumulation and glutamate loss. Changes in keto acid content did not appear to be the cause of amino acid changes. Accumulation of gamma-aminobutyrate was due to acceleration of glutamate decarboxylation and arrest of gamma-aminobutyrate transamination. Changes in enzyme content did not explain the changes in reaction rates in vivo. Most of the aspartate may be converted anaerobically to alanine via oxalacetate and pyruvate.
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Protoplasts were isolated from wheat and spinach leaves and after photosynthesis in the presence of fractionated into a chloroplast and a nonchloroplast fraction. The kinetics of the distribution of labeled metabolities between the fractions indicated carbon flow from the chloroplasts into the cytosol and the vacuole. After 10 min of photosynthesis, more than 90% of the assimilated soluble carbon was outside the chloroplasts. Different metabolite levels indicated intracellular metabolite compartmentation and metabolite gradients. During photosynthesis, gradient coupling facilitated uphill export of triosephosphate from the chloroplasts into the cytosol. The driving force appeared to be an opposite phosphate gradient. Different ratios of phosphoglycerate to triosephosphate inside and outside the chloroplasts indicated the existence of a transenvelope pH gradient. Sucrose appeared to be synthesized outside the chloroplasts and is probably exported into the vacuole. The behavior of malate also suggested transfer into the vacuole. The malate/ aspartate ratio was higher outside the chloroplasts that inside suggesting import of reducing equivalents into the chloroplasts through the dicarboxylate translocator.
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This is about a young man who wished to go to sea like his father and finished up, instead, in photosynthesis. It describes how he served his apprenticeship in England and the United States and how he was then lucky enough to find himself in the laboratory of Robin Hill, one of the all-time greats in this field. It discusses some of the events that led, via mitochondria in castor beans and carboxylating enzymes in Crassulacean plants, to the isolation of fully functional chloroplasts and the manner in which the first polarographic measurements of CO2-dependent O2 evolution contributed to present understanding of the movement of molecules through the chloroplast envelope. It describes some of the problems with materials and apparatus which were commonplace forty years ago and reflects on the advantages of working in foreign places and the pleasures of becoming a member of a truly international community.
Article
The chloroplast envelope is a highly effective barrier against the unidirectional movement of most cations and anions. Still, in illuminated leaf cells there is a rapid exchange of anionic metabolites between chloroplasts and cytosol which profoundly influences extrachloroplast metabolism.
Chapter
Photosynthesis is driven by light. Particularly in low or moderate light, its rate is therefore largely determined by the rate at which photons reach the photosynthetic apparatus. When the rate of photosynthesis is plotted against the rate of arrival of photons (photon flux density or PFD) the relationship (Fig. la) is full of information. The intercept on the vertical axis is a measure of dark respiration. The intercept on the horizontal axis is the “light compensation point” (LCP). This, again, is largely determined by the rate of dark respiration because it is the PFD at which respiratory O2 uptake and photosynthetic oxygen evolution come into balance. In the region between the LCP and 100 µmole quanta.m-2.s-1 the relationship is nearly linear and this initial slope gives the maximum efficiency of energy transduction; usually expressed as quantum yield (O2 evolved per photon absorbed) or its reciprocal, quantum requirement (number of photons required to bring about the evolution of one molecule of oxygen). As will be seen, other information may be derived from the overall rate v PFD relationship by appropriate analysis. Although the importance of this relationship has been long recognised it is only recently that its measurement has become a practical possibility as a routine procedure in non-specialist laboratories.
Chapter
Interaction between photosynthesis and respiration is an enigma. Ever since photosynthetic gas exchange was first measured the question has been posed whether dark respiration continues in the light, and, if so, at what rate (Rabinowitch, 1945; Heath, 1969). The question has some importance for estimation of the growth of plants and of photosynthesis since apparent photosynthetic CO2 fixation (including both photosynthetic CO2 and light-enhanced dark CO2 fixation (see Chap. II.5, this vol.) must be corrected for loss of CO2 by respiratory activity. It is now known that such respiratory activity comprises at least two components, photorespiration which is a light-dependent process, and dark respiration which may continue during illumination. Photorespiration (see Chap. 5.II. 25–28, this vol.) is a light-induced evolution of CO2, and concomitant O2 uptake, involving oxidation of C2-substrates via a complex series of reactions in the chloroplast, peroxisome, and mitochondrion. Dark respiration, on the other hand, involves oxidation of sugars via glycolysis and the tricarboxylic acid (TCA) cycle and the oxidative pentose phosphate (OPP) pathway. The main subject of this chapter is the interaction between photosynthesis and dark respiration during illumination, although the interactions between dark respiration and photorespiration cannot be ignored.
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Evolution of CO2 into CO2-free air was measured in the light and in the dark over a range of temperatures from 15 to 50°. Photosynthetic rates were measured in air and O2-free air over the same range of temperatures. Respiration in the light had a different sensitivity to temperature compared with respiration in the dark. At the lower temperatures the rate of respiration in the light was higher than respiration in the dark, whereas at temperatures above 40° the reverse was observed. For any one species the maximum rates of photosynthesis and photorespiration occur at about the same temperature. The maximum rate for dark respiration generally is found at a temperature about 10° higher. Zea mays and Atriplex nummularia showed no enhancement of photosynthesis in O2-free air nor any evolution of CO2 in CO2-free air at any of the temperatures.
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Rates of true photosynthesis (TPS), apparent photosynthesis (APS) and photorespiration (PR) of sunflower (Helianthus annuus L., Var. Mennonite) leaves were measured in air (21% O2, 300 vpm CO2) at 25° C and 400 μEinsteins m-2 s-1 radiant flux density. The plants were water stressed by application of osmoticum (polyethylene glycol 4000) to the root system. TPS and APS decreased linearly from maxima at-4 bar leaf-water potential (ψ) to become very small and zero respectively at about-18 bar ψ; at smaller potential CO2 was evolved from the leaf. Statistical analysis shows that TPS and APS were more closely correlated with ψ than stomatal conductance (r s-1), because r s-1 changed only in the range-4 to-13 bar but ψ exerted an effect at smaller potential. Photorespiration decreased linearly with stress and at-18 bar was 30% of the control plant rate; ψ and TPS accounted for only part of the variance in PR, both independently and in combination, and r s-1 accounted for little of the variance. Tricarboxylic acid cycle respiration of leaves placed for 20 min in darkness, remained almost constant with changing ψ and r s-1. It was one-third of photorespiration in control plants but increased as a proportion in severely stressed plants. The relative specific activity (RSA) of the CO2 released by PR of wellwatered plants was 90% after 20 min photosynthesis in 14CO2 but decreased to 18% at-18 bar ψ. Therefore, under stress mpre CO2 was derived by respiration from reserve materials and less from immediate photosynthate. Elimination of CO2 production by the glycollate pathway with small oxygen concentration (1.5%), showed that the contribution of TCA cycle respiration to photorespiration was small in unstressed plants but increased at small ψ to almost the same rate as photorespiration. It is concluded that desiccation decreased photosynthesis by decreasing the stomatal conductance to CO2 diffusion and by changing the balance between CO2 assimilation and production of the leaf. As a consequence carbon flux through the glycollate pathway decreased as did the rate of CO2 produced by it. However, TCA cycle respiration in the light increased with stress, so that total photorespiration remained large. The importance of maintaining carbon flux through the glycollate pathway and TCA cycle is discussed.
Article
NAD-specific malate dehydrogenase (EC 1.1.1.37) has been partially purified from mung bean (Phaseolus aureus) hypocotyl mitochondria. ATP inhibited the enzyme competitively with NAD+ at pH 7.2. The degree of inhibition by ATP was also related to the concentration of malate. The Ki for ATP was 0.37 mM at 10 mM at 10 mM malate and 0.85 mM at 20 mM malate. ATP (1 mM) increased the Km for NAD+ from 0.19 mM to 0.61 mM at 10 mM malate and from 0.21 mM to 0.51 mM at 20 mM malate. These results are discussed in relation to the regulation of tricarboxylic acid cycle (TCA cycle) activity in plant mitochondria.
Article
After 10 min illumination of segments of bean (Phaseolus vulgaris L.) or maize (Zea mays L.) leaves in air with 14CO2, the atmosphere was changed to CO2-free O2 or N2 and conversion of photosynthetic products in the light was investigated. The experiments have shown that after the 14CO2 assimilation period the bean leaves contain the pool of weakly fixed 14C (WF-14C) which is converted into stable products during the subsequent period of illumination in CO2-free N2. In O2 atmosphere the WF-14C pool is initially the main source of CO2 evolved. The marked decrease in radioactivity of sucrose and starch during illumination of bean leaves in O2 atmosphere indicates that these compounds were also the source of CO2 evolved in the light. The total amount of previously fixed 14C remained almost on the same level during illumination of maize leaves in N2 as well as in O2. However, oxygen changed the distribution of 14C in photosynthetic products, which is suggested to be the consequence of the photorespiration process in maize.
Article
Leaves of dwarf bean and blackcurrant were analysed for their content of some of the acids of the tricarboxylic acid (TCA) cycle after treatment with lime sulphur in order to correlate any changes with the increase in respiration previously reported.In the dwarf bean, a rapid initial decrease in succinic acid was followed by a large increase. This suggested that there was some blocking of the succinic dehydrogenase by the toxic agent. However it is probably important to consider the contribution of the hexose monophosphate (HMP) shunt in the increased respiration and associated metabolic changes as it has been shown that many disruptive agents, both chemical and biological, favour respiration by the HMP rather than the TCA route in plants.
Article
This chapter discusses the bioenergetic and ultrastructural changes associated with chloroplast development. During development, large amounts of lipids, pigments, proteins, and nucleic acid are synthesized and transformed, requiring expenditure of energy. The chapter discusses these biosynthetic demands in relation to ultrastructure, the appearance of photophosphorylation, and the various control mechanisms involved in the regulation of these events. It also describes the plastid development in different systems. The semicrystalline structure is remarkable in appearance and phototransformation. The chapter outlines the storage reserves and mobilization during plastid development in different systems. The claims of ultrastructural associations between organelles, especially plastids and mitochondria, are fraught with problems. Close appressions of plastids and mitochondria are often observed in all groups of algae. The polypeptide composition of the plastid envelope membranes differs from that of the mitochondrial membranes, the endoplasmic reticulum, and the internal thylakoids. The chapter discusses the formation of pigments, lipids, and the photochemical systems during chloroplast morphogenesis. Various aspects of chloroplast development are directly or indirectly initiated, maintained, or terminated by light and hormones.
Article
Chromatographic separation of an extract of organic acids on a Dowex-l column in the formiate cycle was used to study the content of several organic acids in pea plants, cultivated either in light or in darkness. Concentration changes of the individual acids in the course of growth indicate that the citrate cycle is blocked in the cotyledons of plants grown in light in the period around the 15th day of growth, probably at the site of succinic dehydrogenase (succinic and lactic acids accumulate and the content of citric and malic acids is exhausted). There is no inhibition in the cotyledons of etiolated plants. In vegetative organs, the concentration of the majority of the acids studied is lower than in cotyledons, probably because synthetic processes prevail over degradation processes in these organs. It seems that other processes besides the citrate cycle participate in malate synthesis in pea plants.
Article
Fettspeichernde Samen enthalten im allgemeinen nur C18- Fettsäuren, die bis zur Stufe der Linolsäure desaturiert sind. Zu den Ausnahmen gehören u. a. die Samen von Cruciferen, Leguminosen und Linaceen. Ihre Embryonen entwickeln vor allem in den ersten Reifestadien photosynthetisch aktive Chloroplasten mit einem hohen Gehalt an Linolensäure. Für die Züchtung von Rapspflanzen (Cruciferen) mit einem verminderten Linolensäure-Gehalt im Samenöl ist daher auf Formen auszulesen, in deren Samen während der Reife eine schwächere Ausbildung oder eine frühere Reduktion der Chloroplasten erfolgt.Influence of Chloroplasts on the Formation of Unsaturated Fatty Acids in Maturing RapeseedsIn general, seeds that store fats, contain only C18-fatty acids which are desaturated upto linoleic step. Exceptions to this general pattern are the seeds of cruciferae, legumenae and linaceae. Embryos of these seeds develop, especially during the initial stages of maturation, photosynthetically active chloroplasts with a high content of linolenic acid. Therefore, for the breeding of rapeseed plants (cruciferae) having low linolenic acid content in the seed oil, one has to select either such seeds in which very little chloroplasts are formed during maturation, or seeds in which chloroplasts are reduced at an early stage.
Article
The biochemical evidence concerning the rate of production of CO2 by dark respiratory pathways in illuminated green plant cells is reviewed. Pyruvate decarboxylation and the TCAC are important sources of biosynthetic intermediates in the light as well as in the dark. The rate of CO2 release required to synthesize these carbon skeletons from pyruvate in the light approaches the rate of dark CO2 production. Oxidative phosphorylation, the second important role of the TCAC in the dark, is supplemented and partly replaced by photophosphorylation in the light, and some sort of ‘physiological uncoupling’ may be required to stop the rate of generation of reductant in the TCAC in the light from exceeding the rate of its consumption in oxidative phosphorylation. The decarboxylations of pyruvate and TCAC metabolism occur, in the light as in the dark, in the mitochondria (except possibly in Acetabularia, where they may also occur in the choroplasts). All the decarboxylations produce unhydrated CO2. The oxidative PPP has no obligatory role to play in carbon skeleton synthesis, and it is supplemented in its dark rôle of NADPH2 synthesis by photoproduction of reductant when the cell is illuminated. Evidence as to whether light stimulates or inhibits the oxidative PPP is contradictory. This pathway occurs in at least some illuminated green cells; the decarboxylation reaction produces unhydrated CO2, and occurs in both the cytoplasm and the chloroplast. The situation with regard to carbon flux through the dark respiration pathways in various groups of plants is considered quantitatively. Even without taking into account the contribution from light-dependent pathways of CO2 production (photorespiration), it would appear that the absence of net CO2 release to a CO2-free gas stream in the light in a green cell or tissue cannot entirely be attributed to inhibition of respiration. The absence of CO2 release must also reflect the capacity of the tissue for reassimilation of endogenous CO2.
Article
The rate of carbon dioxide exchange in both light and darkness by detached tobacco leaves placed at various oxygen concentrations was measured by an Infra-Red CO2 Analyzer and a Clark oxygen electrode. It was observed that during illumination oxygen had two different effects. One was to stimulate carbon dioxide evolution and the other to inhibit carbon dioxide absorption. Concentration of carbon dioxide at compensation point was found to be a linear function of oxygen concentration and this has been explained as due mainly to an increased evolution of carbon dioxide. Such an evolution during illumination has been called photorespiration. Increased concentrations of oxygen also had a stimulating effect on the magnitude of the initial post-illumination burst of carbon dioxide in darkness, but no effect on the subsequent steady rates. These data have been explained as due to the suspension of regular respiration in darkness and its replacement by a different process, tentatively called photorespiration. A second effect of oxygen was to reduce the efficiency (called “carboxylation efficiency”) with which a leaf was able to remove carbon dioxide from the atmosphere.
Article
In the presence of large accumulations of protochlorophyllide, derived from exogenous δ-aminolevulinic acid, chlorophyll synthesis in excised leaves of two varieties of barley was less than in untreated leaves. In oat leaves the accumulated protochlorophyllide, from exogenous δ-laminolevulinic acid, stimulated chlorophyll synthesis to above the control level. — These relationships could only be demonstrated when phtodestruction of pigments was minimised by the use of flash illumination (2 milliseconds every 3 minutes). — These was no evidence from in vivo absorption spectra that the pigments in the barley leaves were different to those in leaves studied by other workers. However, the presence of the accumulated protochlorophyllide appeared to prevent the shift of the chlorophyll absorption maximum from 673 nm to 677 nm. — Possible mechanisms of inhibition are discussed.
Article
The respiratory uptake or photosynthetic evolution of oxygen by mesophyll protoplasts of pea (Pisum sativum L. cv. Arkel) were monitored during successive short. (3–5 min) cycles of darkness and illumination. The rate of respiration was nearly doubled after 3–4 short periods of illumination while there was a 15–20% enhancement in photosynthesis with cycles of illumination and darkness preceding illumination. Such interaction between photosynthesis and respiration was statistically significant when bicarbonate was present in the reaction medium. The inhibitors of photosynthesis [3(3,4–dichlorophenyl)-l,l-dimethylurea (DCMU), glyceraldehyde] decreased respiration after periods of illumination, whereas inhibitors of respiratory electron transport (Rotenone, antimycin A, NaN3) suppressed photosynthesis, as well. We suggest that a rapid beneficial interaction exists between photosynthesis and respiration in protoplasts, even during short cycles of light and darkness.
Article
Methods of estimating CO2 release from illuminated green tissues are considered and the rate of decarboxylation deduced from biochemical evidence of substrate turnover is compared with the observed rate of CO2 release; the difference between these values is reassimilation (Table 1). In angiosperms with the PGA pathway, in air, reassimilation is never complete and ranges from o to 0.6 of the endogenous CO2 production. This implies that the resistance to refixation is as great or greater than the resistance to loss of endogenous CO2 to the medium and is in accord with the relative values of these resistances based on other evidence. In law oxygen environment less glycolate is produced and hence less photorespiratory CO2; the resistance to refixation is smaller and so fractional reassimilation in increased. Unicellular algae have the PGA pathway and some appear to have more efficient reassimilation than the corresponding angiosperms.
Article
Single attached leaves of sunflower (Helianthus annus L. "Mennonite") were supplied (14)CO(2) of constant specific radioactivity in gas mixtures containing various CO(2) and O(2) concentrations. The (14)CO(2) and CO(2) fluxes were measured concurrently in an open system using an ionization chamber and infrared gas analyzer.The rate of photorespiration (5.7 +/- 0.3 mg CO(2).dm(-2).(-1)) during photosynthesis in 21% O(2) at 25 C and 3,500 footcandles was over three times the rate of dark respiration and was independent of CO(2) concentrations from 0 to 300 mul/l. The steady rate of CO(2) evolution into CO(2)-free air was about 30% lower. Low oxygen (1%) inhibited both (14)CO(2) and CO(2) evolution, both during photosynthesis and in CO(2)-free air in the light.At 300 mul/l CO(2) apparent photosynthesis was inhibited 41% by 21% O(2). Two-thirds of the inhibition was due to the inhibition of true photosynthesis by oxygen and one-third due to the stimulation of photorespiration. At 50 mul/l CO(2), where the percentage inhibition of apparent photosynthesis by 21% oxygen was 92%, photorespiration accounted for two-thirds of the total inhibition.The rate of (14)CO(2) uptake by the leaf decreased about 30 seconds after the introduction of (14)CO(2), indicating that (14)CO(2) was rapidly evolved from the leaf. The rate of (14)CO(2) evolution increased rapidly with time, the kinetics depending on the CO(2) concentration. The high specific radioactivity of the (14)CO(2) evolved during photosynthesis or in the early period of flushing in CO(2)-free air showed that the substrate for light respiration was an early product of photosynthesis. From the measurement of (14)CO(2) and CO(2) evolution into CO(2)-free air over a longer time period it was apparent that at least three compounds, each of decreased (14)C content, could supply the substrate for light respiration.Based on a consideration of the specific radioactivity of (14)CO(2) evolved under a variety of conditions, it is suggested that total CO(2) evolution in the light or photorespiration is composed of two processes, dark respiration and light respiration. Light respiration is a process that only occurs in the light, persists for some time on darkening, and metabolizes substrates that are quite different from those of dark respiration.
Article
The effects of various mineral salts have been studied, both singly and in combination, on the uptake and distribution of photosynthetically incorporated carbon-14 in Chlorella. When magnesium sulphate, potassium nitrate, and potassium phosphate were combined in the same concentrations as are found in the normal nutrient solution, the uptake of carbon-14 was increased and more radioactivity was incorporated into the amino-acids, with a decrease in the percentage of radioactivity in the sugar phosphates when compared to the controls in distilled water. The most striking effect of the addition of just one salt was obtained with salts containing nitrogen, particularly ammonium chloride, which in one experiment increased the radioactivity found in the amino-acids from 9.9 per cent. (expressed as percentage of the total soluble radioactivity found in this group of compounds) in the controls to 57 per cent., while the radioactivity in the sugar phosphates fell from 64 to 7 per cent., correspondingly. The total uptake of carbon dioxide by the cells supplied with ammonium chloride was three times as great as that in the controls in distilled water. The relevance of these findings to the methodology involved in experiments dealing with the path of carbon in photosynthesis is discussed, with particular emphasis upon the concept of a ‘steady state’ and how it is thought this may best be achieved.
Article
Die Trennung des Phänomens der Photosynthese grüner Pflanzen in eine Lichtreaktion und die vom Licht unabhängige Reduktion der Kohlensäure werden diskutiert. Die Reduktion der Kohlensäure und das Schicksal des assimilierten Kohlenstoffs wurden untersucht mit Hilfe der Spurenmethode (Markierung der assimilierten Kohlensäure mit C14) und der Papierchromatographie. Ein Reaktionszyklus wird vorgeschlagen, in dem Phosphoglyzerinsäure das erste isolierbare Assimilationsprodukt ist. Analysierung des Extraktes von Algen, die in einem stationären Zustand für längere Zeit radioaktive Kohlensäure assimilierten, lieferte weitere Auskunft über den vorgeschlagenen Zyklus und gestattete, die am Zyklus beteiligten Mengen einiger Substanzen ungefähr zu bestimmen. Die frühere Vermutung, dass Licht den Respirationszyklus beeinflusst, wird bestätigt. Die Möglichkeit der Mitwirkung von α-Liponsäure (α-lipoic acid) oder einer verwandten Substanz, bei diesem Effekt und im Photosynthesezyklus, wird erörtert.
Article
Kinetic studies have been made of the rates of appearance of 14C in individual compounds formed by Chlorella pyrenoidosa during steady state photosynthesis with 14CO2. These rates have been compared with rates of CO2 and 14C disappearance from the gas phase during the same experiments. 1.The following results were obtained:2.1. After the first few seconds, the rate of appearance of 14C in compounds stable to drying on planchets at room temperature is 95 to 100% of the rate of uptake of carbon from the gas phase.3.2. After the first few seconds, the rate of appearance of carbon in compounds isolable by usual methods of paper chromatography constitutes at least 73 to 88% of the rate of uptake of carbon from the gas phase. Compounds formed from the carbon reduction cycle via the carboxylation of ribulose diphosphate account for a least 70 to 85% of the uptake, while carboxylation of phosphoenolpyruvic acid appears to account for at least another 3%.4.3. The induction period in the appearance of 14C in stable compounds may be due to a reservoir of intracellular CO2 and HCO3− or to some other volatile or unstable compound. If so, this reservoir contains no more than 1.5 μmoles of carbon, corresponding to about 7 sec carbon fixation in the experiment in which it was measured.5.4. No other carboxylation reactions, such as the carboxylation of γ-aminobutyric acid, could be observed. The rate of labeling of glutamic acid after 5 min of exposure of the algae to 14CO2 reached a maximum rate of about 5% of the total uptake rate, but this labeling appears to be due to conversion of labeled intermediates formed from the carbon reduction cycle or phosphoenolpyruvic acid carboxylation.6.5. The in vivo carboxylation of ribulose diphosphate in the light appears to be followed by conversion of the product to one molecule of phosphoglyceric acid, containing the newly incorporated 14CO2 and one molecule of some other (kinetically distinguishable) three carbon compound. This reaction would be different from the one reported for the isolated enzyme system and the in vivo reaction in the dark, which produces two molecules of 3-phosphoglyceric acid.
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