The Effects of Leaf Age and Senescence on the Distribution of Carbon in Lolium temulentum

Assimilate distribution in leaves of Lolium temulentum was establishedby root absorption of [¹⁴C]sucrose and after exposure to ¹⁴CO₂.Age determined the amount of carbon assimilated, with more labelbeing incorporated during expansion than at maturity. Duringsenescence ¹⁴C assimilation was much lower. Ethanol-solubleextracts from various tissues of root-labelled plants containedmost of the radioactivity chiefly in basic and acidic compounds.The neutral fraction was composed predominantly of sucrose. Sucrose was comparably labelled in leaves from plants fed equalamounts of either [¹⁴C]sucrose, glucose, or fructose and onlytraces of labelled monosaccharides appeared in extracts. Radioactive sucrose was translocated rapidly from mature leaveswhereas, in the expanding leaf, carbon incorporation was directedtowards growth and the greater proportion of label present atligule formation was in ethanol-insoluble material. Induced senescence, of a mature leaf fed during expansion, produceda rapid loss from the pool of insoluble ¹⁴C. This was accompaniedby a reduction in the contents of chlorophyll and soluble proteinand an accumulation of amino acids. The onset of senescencecaused changes in leaf sugar levels which were correlated withincreased rates of respiration.     

Metabolism of Exogenous Malonate by Coffee Leaf Tissue

When [l-¹⁴C]-malonate was supplied to discs cut from matureleaves of Coffea arabica, ¹⁴CO₂ was released (approximately12% of the total CO₂ respired) and organic acids of the Krebscycle, uronic acids, sugars and amino acids became radioactive.There was no incorporation of MC into either lipids or phenoliccompounds. The formation of glucose from malonate has not beenobserved in other studies with plant tissues. The synthesisof labelled glucose together with an active pentose phosphatepathway that is stimulated by malonate explains the accumulationof radioactive phosphogluconate in the leaf discs. Tentativeproposals are made for pathways to account for the results obtained. Key words: Coffee leaves, Malonate metabolism, Pentose phosphate pathway     

Effect of leaf age on light and dark 14CO2 fixation in a CAM plant, Bryophyllum calycinum

Leaves of different ages from B. calycinum were exposed to ¹⁴CO₂in light during day and night. The labelling pattern on thechromatogram differed with leaf age. Young leaves had similarpatterns to those of C3 plants during both day and night. Matureleaves showed high incorporation of ¹⁴C into C4 acids, especiallyat night. In contrast, no significant difference with leaf agewas observed in the pattern of dark ¹⁴CO₂ fixation products.Study of the enzyme activity and the content of titratable acidat each leaf age suggested that high incorporation of ¹⁴C inC4 acids during the night was due to the simultaneous absorptionof CO₂ by both enzymes RuDPcarboxylase and PEPcarboxylase. (Received November 24, 1977; )     

Dynamics of Carbon Photosynthetically Assimilated in Nodulated Soya Bean Plants under Steady-state Conditions 2. The Incorporation of 13C into Carbohydrates, Organic Acids, Amino Acids and some Storage Compounds

Nodulated soya bean (Glycine max L.) plants at the early floweringstage were allowed to assimilate ¹³CO₂ under steady-state conditions,with a constant ¹³C abundance, for 8 h in the light. The plantswere either harvested immediately or 2 d after the end of the¹³CO₂ feeding, divided into young leaves (including flower buds),mature leaves, stems+petioles, roots and nodules; the ¹³C abundancein soluble carbohydrates, organic acids, amino acids, starchand poly-ß-hydroxybutyric acid was determined witha gas chromatography-mass spectrometry. The rapid turnover of ¹³C in the sucrose pools observed in allorgans of the plants showed that sucrose was the principal materialin the translocation stream of primary products of photosynthesis.At the end of the ¹³CO₂ exposure, sucrose in the mature leavesas the major source organs and in the stems+petioles was labelledwith currently assimilated carbon to about 75 per cent, whereasa much higher labelling of sucrose was found in the roots andin the nodules. This suggests the existence of two or more compartmentedpools of sucrose in mature leaves and also in stems+petioles. The relative labelling patterns of individual organic acidsand amino acids were similar in various plant organs. However,the rapid turnover of succinate and glycine was characteristicof nodules. Treatment with a high concentration of nitrate inthe nutrient media increased the turnover rate of amino acidcarbon in shoot organs and roots, while it markedly decreasedthe labelling of amino acids in nodules. The cyclitols, exceptfor D-pinitol, were significantly labelled with assimilated¹³C in mature leaves, but in nodules, the labelling was verymuch less. In the nodules, which were actively fixing atmospheric nitrogen,a large proportion (80–90 per cent) of currently assimilatedcarbon was found as sucrose and starch at the end of the ¹³CO₂feeding. This was also true of the roots. On the other hand,in young growing leaves, the distribution of currently assimilatedcarbon into sucrose, starch and other soluble compounds wasmuch less. This suggests that a large amount of carbon assimilatedby and translocated to young leaves was used to make up structuralmaterials, mainly protein and cell wall polymers synthesis,during the light period. Glycine max L., soya bean, ¹³CO₂ assimilation, carbon metabolism in nodules     

Further studies on the metabolism of glucose in the process of "glucose-bleaching" of Chlorella protothecoides

Previous studies have demonstrated that when cells of Chlorellaprotothecoides are incubated in a medium containing glucosebut no nitrogen source, they are profoundly bleached with degenerationof chloroplast structure and photosynthetic activity. When anitrogen source (urea) is added to the glucose medium, bleachingof algal cells is greatly suppressed. In this work the metabolismof glucose in the process of glucose-induced bleaching was studiedusing ¹⁴C-glucose as tracer. Changes in algal cell activityfor ¹⁴CO₂-evolution and ¹⁴C-incorporation into various cellularsubstances from ¹⁴C-glucose were followed. Most conspicuouswere increases in cellular activities for assimilating ¹⁴C-glucoseinto lipids (fatty acids) and glucose polymer. When urea wasadded to the glucose medium, the incorporation of ¹⁴C by algalcells into fatty acids was greatly reduced, while the assimilationof ¹⁴C into glucose polymer was increased. These and previous observations suggest that the formation oflarge amounts of lipids (fatty acids) probably is causally relatedto the induction of algal cell bleaching. (Received March 5, 1969; )     

Morphological and Physiological Responses of Echinoderm Larvae to Nutritive Signals

SYNOPSIS. Echinoid larvae have been found previously to developshorter arms as phytoplankton concentrations increase. In thepresent study, the skeletal dimensions of larvae of the seaurchins Lytechinus pictus and Strongylocentrotus purpuratuswere measured after exposure to dissolved organic compoundsin seawater. The presence of glucose or individual amino acids(at 1 or 2 µM), a mixture of 16 different amino acids(100 nM each), or algal exudate resulted in larvae with shorterarms (by 12 to 88%), relative to larvae in seawater with noadditions. Larvae exposed to mixtures of amino acids also hadchanges in the constituents of their internal free amino acidpools (as determined by HPLC). For another echinoid, Dendrasterexcentricus, amino acid transport (T, from 500 nM) by individuallarvae (n = 47) scaled to arm length (L) as follows: T = 2.06L^0.81(0.81 ± 0.26,95% confidence intervals). Mass (M) andmetabolic rate (MR) did not scale in the same manner (as transport)to arm length for larvae of the echinoid Centrostephanus coronatus(MR = 148L^1.51; M = 1.7L^2.01). These characteristics may scaleto arm length independently from transport rate under certainconditions. Larvae of echinoderms respond morphologically to"signals" in their environment that may indicate the availabilityof dissolved and paniculate nutrients. This in turn will haveconsequences for their ability to take up and metabolize thesenutrients. The response may be mediated by surface receptorsfor dissolved organic compounds, or by changes in the sizesof intracellular substrate pools.     

Formation of putrescine from ornithine and arginine in tobacco plants

Putrescine was formed when both DL-ornithine-2-¹⁴C and L-arginine-U-¹⁴Cwere injected into tobacco plants. The incorporation rate ofthese amino acids into putrescine was higher in potassium orsulfur deficient plants than in normal plants. Degradation ofputrescine-1,4-¹⁴C injected into tobacco plants was inhibitedby a potassium or sulfur deficiency. Increased accumulationof putrescine in potassium or sulfur deficient plants may bepartly dependent upon this inhibition in the degradation ofputrescine. Detached leaves and roots were also able to formputrescine from DL-ornithine-2-¹⁴C and L-arginine-U-¹⁴C. (Received December 13, 1968; )     

METABOLISM OF ACETATE-1-14C IN EXCISED LEAVES FROM POTASSIUM DEFICIENT RICE SEEDLINGS

Excised leaves from one-month old rice seedlings grown in potassiumdeficient nutrient solution were fed with acetate-l-¹⁴C for20 to 180 min. Rate of respiration of those leaves was significantlyhigher than that of control leaves supplied with sufficientpotassium. Incorporation of UC into organic acid and sugar fractionswere greater in potassium deficient leaves than in normal leaves.The reverse was true as for the incorporation into amino acidfraction. In comparison with normal leaves, potassium deficientleaves acquired much ¹⁴C in succinic acid and glutamine. Therewere distinct differences in the labeling pattern of citrateand in the order of magnitude of specific activities among theorganic acids included in the TCA cycle. From these results,it was assumed that the increase of respiratory rate causedby the potassium deficiency was related to accelerated operationof the TCA cycle. The decreased rate of conversion of organicacids to amino acids and the accumulation of glutamine werealso concluded. (Received April 13, 1967; )     

Contrasting effects of substrate and grazer manipulations on picoplankton in oceanic and coastal waters off Brazil

In two contrasting regions off the coast of Brazil, picoplankton(<1 µm) responses to removal of larger grazers andto the additions of glucose and amino acids were determined.Effects of glucose and amino acid additions (1 µM) onparticulate nitrogen and chlorophyll a concentrations, and onrates of NH₄⁺ uptake and regeneration, were observed after 5h pre-incubation. In the oceanic waters, removal of the >1µm fraction had no significant effect on the chlorophylla of the picoplankton after 5 h. However, the addition of glucosestimulated both uptake and regeneration by a mean of 27%, andthe addition of arginine led to significant decreases in therates of NH₄⁺ uptake and regeneration. In contrast, in the coastalwaters, significant increases in chlorophyll and particulatenitrogen concentrations were found after 5 h incubation in boththe amended samples and in the controls, and mean rates of NH₄⁺uptake and regeneration were affected to a lesser degree bythe additions of either glucose or amino acids than in the oceanicwaters. The oceanic responses were suggestive of carbon limitationof heterotrophic bacteria. In the coastal region, on the otherhand, the supply of organic carbon and nitrogen was likely tohave been sufficient to meet the nutritional requirements ofthe heterotrophic bacteria and cyanobacteria. Grazing by largerorganisms on the picoplankton appeared to play a more significantrole in the nitrogen cycle in the coastal waters than in theoceanic waters.     

Arginine metabolism by pumpkin seedlings. Separation of plant extracts by ion exchange resins

Arginine-U-¹⁴C was injected into the cotyledons of 7-day oldpumpkin seedlings. At most, 24% of the administered ¹⁴C wastransported to the axis tissue. The amounts of arginine incorporatedinto cotyledonary protein suggests that turnover was occurringat a rapid rate. Arginine was extensively metabolized, and after96 hr 50% of the administered ¹⁴C had been released as ¹⁴CO₂.The remaining label was primarily in unmetabolized arginine,protein or transported to the axis tissue with little labelin other amino acids. The results suggest that the carbon fromarginine is incorporated into protein or catabolized to CO₂while the carbon for new amino acid skeletons is derived fromsugar. A simple, reproducible method for the quantitative fractionationof plant extracts or hydrolysates of insoluble plant materialinto basic amino acids, acidic amino acids, neutral amino acids,organic acids and sugars was reported. (Received September 10, 1968; )     

Mobilization of Reserves and Synthesis of Sterols and Triterpenes in Seedlings of Two Canarian Euphorbia Species

Seedlings from Euphorbia canariensis and Euphorbia lambii weregrown in the dark at 25 °C. Protein and triglyceride contentas well as levels of sugars and amino acids in the endospermwere determined during endosperm depletion. In the endospermof Euphorbia canariensis, relatively low levels of amino acids(up to 1 µmol.endosperm^–1) were found of which glutamine/glutamateaccounted for 40% at the stage of radicle emergence. High levelsof amino acids (up to 4 µmol.endosperm^–1) comparedwith sugars (up to 2 µmol sucrose.endosperm^–1) weredetected in the endosperm of Euphorbia lambii. Arginine wasthe main component (28 µmol%) of the amino acids in thistissue. In both species amino acid composition changed graduallyduring endosperm depletion. Cotyledons retained their ability to absorb a variety of watersoluble substrates after removal of the endosperm. ¹⁴C from[U-¹⁴C]sucrose was effectively incorporated into the triterpenesof the laticifers and to a lesser extent into the sterols ofthe seedling. The highest incorporation values were found inyoung seedlings about 2 d after the emergence of the radicle.Seedlings of this age also showed high incorporation rates of¹⁴C from labelled alanine, serine, threonine, valine, leucineand isoleucine into both triterpenols and sterols, but no generalconclusions about metabolic channelling in lipid synthesis couldbe made. Endosperm, Euphorbia canariensis L. Euphorbia lambii Svent., sterols, triterpenols, amino acids, laticifer, biosynthesis     

Effect of growth temperature on carbon isotopic ratios in barley, pea and rape

Barley, pea and rape were grown in controlled environments atday/night temperatures ranging from 13/4 to 35/27?C. Shoot tissuewas analyzed for changes in the natural abundance ratio of ^l3C/¹²C.More¹³C was incorporated during growth at low temperatures thanat higher temperatures, but the change in isotopic ratio wasnot linear over the temperature range employed. ³Present address: Faculty of Agriculture, University of Ceylon,Peradeniya, Ceylon. (Received September 13, 1972; )     

Isolation and some properties of copper-binding proteins found in a copper-resistant strain of yeast

Copper-binding proteins were extracted from a copper-resistantstrain of Saccharomyces cerevisiae which was obtained by repeatedsubculturing in a copper-containing medium. They were separatedinto three types through purification steps such as salt fractionation,gel filtration and preparative polyacrylamide gel electrophoresis.They resembled each other in amino acid composition. Acidicamino acids, lysine, serine, glycine and half-cystine constituteda large part of the protein, with a small amount of hydrophobicamino acids. Aromatic amino acids and methionine were almostabsent. The molecular weight of the components was estimatedto be about 10,000 by Sephadex gel filtration and electrophoresison polyacrylamide gel (slope method). Absorption spectra ofthe components exhibited a broad band at 275 nm, but none inthe visible region, thus resembling that of copper-thionein.Moreover, the absorption band at 275 nm changed markedly onaddition of Ag⁺, Hg2⁺, CN^– or H₂O₂, which are well knownas thiol reagents. These components were abo produced in theparent cells, if they could grow in a copper-containing medium.Based the results of experiments using various culture conditionsand some other yeast species, a possible role of the componentsis discussed. (Received July 13, 1976; )     

Dynamics of simple carbon compounds in two forest soils as revealed by soil solution concentrations and biodegradation kinetics

Simple compounds in soil such as organic acids, amino acids and monosaccharides are believed to be important in regulating many aspects of terrestrial ecosystem functioning (e.g. C cycling, nutrient acquisition). Understanding the fate and dynamics of these low molecular weight (MW) compounds is therefore essential for predicting ecosystem responses to disturbance. Our aim was to quantify the amounts of these compounds in two podzolic forest soil profiles (O, E, Bs and C horizons) and to quantify their contribution to total soil respiration. The total concentration of organic acids, amino acids and monosaccharides in soil solution comprised on average 15?±?10% of the total dissolved organic C (DOC), with declining concentrations in the deeper soil layers. Dissolved organic N (DON) was the dominant form of N in soil solution and free amino acids contributed to 34% of this pool. The mineralization behaviour of glucose and galactose was described by parabolic (Michaelis–Menten) type kinetics with V _max and K _M values in the range of <1–250 μmol kg^?1 h^?1 and 15–1,100 μM, respectively. Assuming that (1) microbially mediated substrate turnover follows Michaelis–Menten kinetics, and (2) steady state soil solution concentrations, we calculated the rate of CO₂ efflux attributable to the mineralisation of the three classes of low MW compounds. Our results indicated that in the O horizon, the turnover of these substrates could comprise ~100% of the basal, heterotrophic, soil respiration. In contrast, in the deeper mineral soil <20% of total soil respiration could be attributable to the mineralization of these compounds. Our compound-specific approach has identified the main substrates contributing to soil respiration in forest topsoils. However, our results also suggest that soil respiration in subsoils may be attributable to compounds other than organic acids, amino acids and monosaccharides.     

Studies on Translocation of Metabolites in the Xylem of Grapevine Shoots

Fluid extracted from grapevine shoots by suction contained upto 0.2 per cent (w/v) of sugar. The total sugar concentrationand the relative concentrations of sucrose, glucose, and fructosechanged along the length of a shoot. Whereas sucrose is absentfrom or present only in traces in fluid bleeding from cut stumps,it was found to be a major constituent in fluid extracted fromsections of wood by suction. ¹⁴C-glucose administered to thewood of an excised shoot moved up in the transpiration stream,moved laterally, into the bark, and was partly converted tosucrose and fructose. When ¹⁴CO₂ was supplied to a leaf of anintact vine, radioactive sugars were found in the fluid afterwardsextracted from the wood by suction. When a ring of bark wasremoved from the stem at a point above the ¹⁴CO₂-treated leafmost of the ¹⁴C in the bark, wood, and extracted fluid belowthe ring was in the form of sugar but ¹⁴C in tissues above thering was mainly in organic acids and amino acids. It is suggestedthat a barrier within the wood prevents diffusion of sugarsinto the transpiration stream, and that the fluid extractedfrom the wood by suction cannot be regarded as ascending sapsince it contains considerable amounts of non-moving material.     

Sorting of metabolic pathway flux by the plasma membrane in cerebrovascular smooth muscle cells

We used-escin-permeabilized pig cerebral microvessels (PCMV) to study theorganization of carbohydrate metabolism in the cytoplasm of vascularsmooth muscle (VSM) cells. We have previously demonstrated (Lloyd PGand Hardin CD. Am J Physiol Cell Physiol 277: C1250-C1262,1999) that intact PCMV metabolize the glycolytic intermediate[1-¹³C]fructose 1,6-bisphosphate (FBP) to[1-¹³C]glucose with negligible production of[3-¹³C]lactate, while simultaneouslymetabolizing [2-¹³C]glucose to[2-¹³C]lactate. Thus gluconeogenic andglycolytic intermediates do not mix freely in intact VSM cells(compartmentation). Permeabilized PCMV retained the ability tometabolize [2-¹³C]glucose to[2-¹³C]lactate and to metabolize[1-¹³C]FBP to[1-¹³C]glucose. The continued existence ofglycolytic and gluconeogenic activity in permeabilized cells suggeststhat the intermediates of these pathways are channeled (directlytransferred) between enzymes. Both glycolytic and gluconeogenic flux inpermeabilized PCMV were sensitive to the presence of exogenous ATP andNAD. It was most interesting that a major product of[1-¹³C]FBP metabolism in permeabilized PCMV was[3-¹³C]lactate, in direct contrast to ourprevious findings in intact PCMV. Thus disruption of the plasmamembrane altered the distribution of substrates between the glycolyticand gluconeogenic pathways. These data suggest that organization of theplasma membrane into distinct microdomains plays an important role insorting intermediates between the glycolytic and gluconeogenic pathwaysin intact cells.

     

Dynamics of Carbon Photosynthetically Assimilated in Nodulated Soya Bean Plants under Steady-state Conditions 3. Time-course Study on 13C Incorporation into Soluble Metabolites and Respiratory Evolution of 13CO2 from Roots and Nodules

Well-nodulated soya bean (Glycine max L.) plants were allowedto assimilate ¹³CO₂ for 10 h in the light, under steady-stateconditions in which CO₂ concentration and ¹³C abundance wereboth strictly controlled at constant levels. The respiratoryevolution of ¹³CO₂ from roots and nodules and ¹³C incorporationinto various metabolic fractions were measured during the ¹³CO₂feeding and subsequent 48 h chase period. CO₂ respired from nodules was much more rapidly labelled with¹³C than that from roots. The level of labelling (percentageof carbon currently assimilated during the ¹³COM₂ feeding period)of CO₂ respired from nodules reached a maximum of about 87 percent after 4 h of steady-state ^l3CO₂ assimilation and thereafterremained fairly constant. The absolute amount of labelled carbonevolved by the respiration of the nodules during the 10 h ¹³CO₂feeding period was 1·5-fold that of root respiration.These results demonstrated that the currently assimilated (labelled)carbon was preferentially used to support nodule respiration,while root respiration relied considerably on earlier (non-labelled)carbon reserved in the roots. Sucrose pools were mostly composed of currently assimilatedcarbon in all tissues of the plants, since the levels of labellingaccounted for 86–91 per cent at the end of the ¹³CO₂ feeding.In the nodules, the kinetics and levels of sucrose labellingwere in fairly good agreement with those of respired CO₂, whilein the roots, the level of labelling of respired CO₂ was significantlylower than that of sucrose. Succinate and malate were highly labelled in both roots andnodules but they were labelled much more slowly than sucroseand respired CO₂. The kinetics and levels of labelling of theseKrebs cycle intermediates resembled those of major amino acidswhich are derived directly from Krebs cycle intermediates. Itis suggested that large fractions of organic acids in noduleswere physically separate from the respiration site. Glycine max L., Soya bean, ¹³CO₂ assimilation, respiratory evolution of ¹³CO₂, carbon metabolism in root nodules     

Glycolytic flux in permeabilized freshly isolated vascular smooth muscle cells

To determine whether channeling of glycolytic intermediates canoccur in vascular smooth muscle (VSM), we permeabilized freshly isolated VSM cells from hog carotid arteries with dextran sulfate. Thedextran sulfate-treated cells did not exclude trypan blue, a dye withmolecular weight of ~1,000. If glycolytic intermediates freelydiffuse, plasmalemmal permeabilization would allow intermediates toexit the cell and glycolytic flux should cease. We incubated permeabilized and nonpermeabilized cells with 5 mM[1-¹³C]glucose at37°C for 3 h. ¹³C nuclearmagnetic resonance (NMR) was used to determine relative [3-¹³C]lactateproduction and to identify any¹³C-labeled glycolyticintermediates that exited from the permeabilized cells.[3-¹³C]lactateproduction from[1-¹³C]glucose wasdecreased by an average of 32% (n = 6) in permeabilized cells compared with intact cells. No¹³C-labeled glycolyticintermediates were observed in the bathing solution of permeabilizedcells. We conclude that channeling of glycolytic intermediates canoccur in VSM cells.

     

Two receptor sites and their relation to amino acid stimulation in the labellar sugar receptor of the fleshfly.

Different effects of treatment with reagents such as N-bromosuccimide (NBS) and 2,4,6-trinitrobenzene sulphonic acid (TNBS) on the responses to glucose and to fructose presented further evidence of the existence of two different receptor sites in the labellar sugar receptor of the fleshfly.A clear differentiation of the two receptor sites was suggested by the effects of treatment with p-chloromercuribenzoate (PCMB) on the response to the mixtures of 1 M glucose with various concentrations of fructose.All six amino acids that can stimulate the sugar receptor cell were shown to react with the furanose site, one of the two sugar receptor sites, according to the effects of PCMB and TNBS treatments. The results are discussed in relation to the basic structure of six amino acids and that of monosaccharides in the furanose form essential for stimulation.