Effects of Chloramphenicol on the Uptake and Incorporation of Amino-acids by Carrot Root Tissue

The uptake of uniformly labelled L-glutamic acid-¹⁴C, glycine-¹⁴C,and L-proline-¹⁴C by carrot root tissue is inhibited by chloramphenicolat a concentration of 2 g./l. L-glutamic acid absorption isaffected to a much greater extent than is the uptake of theother two amino-acids. It is shown that the ¹⁴C from the labelledamino-acids is incorporated into protein of the carrot slices,and that the amount of incorporation from glycine and L-prolineis about six times as large as that from L-glutamic acid undercomparable conditions. Almost all the ¹⁴C detected in the proteinafter feeding labelled L-proline or glycine remains in the amino-acidsupplied; in the case of L-glutamic acid some of the label istransferred to other amino-acid residues, but most of it remainsin glutamic acid. Chloramphenicol is found to inhibit ¹⁴C-incorporationfrom L-proline and glycine in short-term, but not in long-term,experiments whereas incorporation from L-glutamic acid is inhibitedin both long- and short-term experiments. Chloramphenicol alsoinhibits incorporation of ¹⁴C from L-glutamic acid into proteinsof preparations of isolated subcellular particles from carrotroot tissue. Under some conditions, ¹⁴C incorporation into proteinproceeds for some time after the slices have been removed fromthe radioactive solution. Such incorporation is not inhibitedsignificantly by chloramphenicol when ¹⁴C-labelled L-prolineis supplied, but it is reduced by about 50 per cent. when glycineand L-glutamic acid are used. It is shown that chloramphenicolinhibits net protein synthesis in carrot root slices suspendedin aerated solutions. It is concluded that amino-acid incorporationinto the protein of carrot slices proceeds by at least two mechanisms,one of which is related to protein synthesis and is sensitiveto chloramphenicol and the other is unrelated to protein synthesisand insensitive to the drug. Most of the L-proline and glycineincorporation into carrot root tissue protein seems to occurby the chloramphenicol-insensitive mechanism while much of theL-glutamic acid-¹⁴C incorporation seems to take place by thechloramphenicol-sensitive one.     

The metabolism of proline in cotyledons of pumpkin (Cucurbita moschata)

Exogenous proline-U-₁₄C is readily metabolized to glutamate,ornithine, sugars, CO₂, and organic acids, and is incorporatedinto protein by etiolated and green pumpkin cotyledons. As littletranslocation of proline from the cotyledons occur, it was proposedthat in young tissue proline is converted to glutamate, ornithineor sugar which are then readily translocated from the cotyledons.In older tissue some glutamate carbon derived from proline isalso used as an energy source and metabolized to CO₂. As proteinsynthesis is occurring rapidly in these cotyledons, considerableproline is incorporated into new protein. After 10-hr, 15% ofthe absorbed radioactivity still remained as free proline. ¹Present address: Instituto de Ciencias Biologicas, UniversidadeFederal de Vicosa, Vicosa, Minas Gerais, Brasil. (Received February 1, 1974; )     

Ethanolamine Metabolism in Plant Tissues

Ethanolamine is readily metabolized by oat, pea, wheat, apple and carrot tissue preparations. Ethanolamine-1,2 (14)C was incorporated into the lipid fraction, and (14)C activity was distributed in the organic acid, sugar, acid volatile, carbon dioxide and insoluble residue fractions. The distribution varied with the particular tissue. Incorporation into the lipid fraction occurred in tissue homogenates in the absence of ATP by a Ca(++) activated system similar to that reported for animal preparations. The initial step in ethanolamine oxidation involves an amine oxidase. Glycolaldehyde and glyoxylic acid are metabolic intermediates, the former in the conversion of ethanolamine to carbon dioxide. No evidence was obtained for the operation of an ethanolamine transaminase or for the involvement of phosphorylated intermediates in the conversion of ethanolamine to carbon dioxide.     

The Incorporation of 14C-Proline into the Proteins of Growing Cells: II. A NOTE ON EVIDENCE FROM CULTURED CARROT EXPLANTS BY ACRYLAMIDE GEL ELECTROPHORESIS

The method of acrylamide gel electrophoresis has been appliedto the separation of the proteins of growing carrot explantswhich are soluble in a buffer solution at pH 8.3. At least ninedistinct bands were detectable in this way. When carrot tissuehad absorbed ¹⁴C-proline it entered into the composition ofall these bands and, in all except one, it was converted tohydroxyproline to different degrees which characterized theband in question. Thus, in the soluble protein the ¹⁴C-hydroxyproline:¹⁴C-proline ratio varied from 0 to 0.53. The bulk of the proteinin the tissue was insoluble in buffer at pH 8.3; it containedthe bulk of the radioactivity absorbed from proline (84.8 percent.); and its average ¹⁴C-hydroxyproline : ¹⁴C-proline ratiowas the highest of all (1.28). A particulate protein preparation,separated at 25,000 g. from the soluble protein, had an intermediateratio (0.643) of ¹⁴C-hydroxyproline to ¹⁴C-proline. Therefore,there are in cultured carrot explants many distinct proteinmoieties which incorporate ¹⁴C-proline, and they convert itto ¹⁴C-hydroxyproline to very different degrees. The evidenceis consistent with the incorporation of the proline, first intothe various soluble proteins which are electrophoretically separableand subsequently, with progressively greater hydroxylation,into the more insoluble protein that constitutes the bulk ofthe protein of the cell and its organelles. It is, therefore,quite incorrect to say (as some have done) that all of the hydroxyproline-containingprotein is in very close association with the cell wall, forpart of it is present in the cell in soluble and electrophoreticallyseparable forms.     

The Influence of Partial Pressure of Carbon Dioxide upon Carbon Metabolism in the Tomato Leaf

The incorporation of radioactive carbon into various photosyntheticproducts was investigated with tomato plants in atmospherescontaining between 40 and 1400 parts/10⁶ carbon dioxide. A significantlygreater proportion of ¹⁴C entered sucrose and alcohol-insolublematerial at high concentrations of carbon dioxide. Incorporationinto glycine and serine was significantly greater at lower carbon-dioxideconcentrations. The pool size of these intermediates was alsodetermined and it was concluded that in the presence of highpartial pressures of carbon dioxide the flow of carbon fromthe photosynthetic cycle through the C² pathway is decreased.     

Succinate metabolism related to lipid synthesis in the housefly Musca domestica L

The metabolism of succinate was examined in the housefly Musca domestica L. The labeled carbons from [2,3-¹⁴C]succinate were readily incorporated into cuticular hydrocarbon and internal lipid, whereas radioactivity from [1,4-¹⁴C]succinate was not incorporated into either fraction. Examination of the incorporation of [2,3-¹⁴C]succinate, [1-¹⁴C]acetate, and [U-¹⁴C]proline into hydrocarbon by radio-gas-liquid chromatography showed that each substrate gave a similar labeling pattern, which suggested that succinate and proline were converted to acetyl-CoA prior to incorporation into hydrocarbons. Carbon-13 nuclear magnetic resonance showed that the labeled carbons from [2,3-¹³C]succinate enriched carbons 1, 2, and 3 of hydrocarbons with carbon-carbon coupling showing that carbons 2 and 3 of succinate were incorporated as an intact unit. Radio-high-performance liquid chromatographic analysis of [2,3-¹⁴C]succinate metabolism by mitochondrial preparations showed that in addition to labeling fumarate, malate, and citrate, considerable radioactivity was also present in the acetate fraction. The data show that succinate was not converted to methylmalonate and did not label hydrocarbon via a methylmalonyl derivative. Malic enzyme was assayed in sonicated mitochondria prepared from the abdomens and thoraces of 1- and 4-day-old insects; higher activity was obtained with NAD⁺ in mitochondria prepared from thoraces, whereas NADP⁺ gave higher activity with abdomen preparations. These data document the metabolism of succinate to acetyl-CoA and not to a methylmalonyl unit prior to incorporation into lipid in the housefly and establish the role of the malic enzyme in this process.     

Studies of Glycollate Utilization and Some Associated Enzymes of C1 Metabolism in the Endosperm of Ricinus communis L.

Glycollate metabolism in 5-day-old endosperm tissues of Ricinuscommunis L. was examined by feeding micromolar quantities of[2-¹⁴C]glycollate to tissue slices. It was found that glycollatecarbon was rapidly incorporated into glyoxylate, glycine, serine,and carbon dioxide. Only small amounts of ¹⁴C were incorporatedinto the sugars. Changes in the distribution of ¹⁴C with timesuggested that glyoxylate was a primary product and that glycineand serine were secondary products of glycollate metabolism.The results of feeding experiments are interpreted as indicatingthat a glycollate pathway leading to sugar biosynthesis is ofminor importance compared to the rapid utilization of glycollatefor the biosynthesis of glycine and serine. Enzymes necessaryto catalyse the incorporation of glycollate into glycine andserine have been examined in castor-bean endosperm extracts.These included: glycollic acid oxidase, gloxylic acid reductase,glyoxylate transaminase, N¹⁰ formyltetrahydrofolate synthetase,N⁵,N¹⁰-methylenetetrahydrofolate dehydrogenase, and serine hydroxymethyltransferase.     

Participation of farnesol in the biosynthesis of ipomeamarone

Farnesol-2-¹⁴C was readily incorporated into ipomeamarone, oneof the furanoterpenoids produced in sweet potato infected withthe black rot fungus, Ceratocystis fumbriata. This was demonstratedby isolating labeled ipomeamarone and analyzing its radioauto-gramby silica gel thin layer chromatography of the extracts solublein chloroformmethanol (1: 1 , v/v), after farnesol-2-¹⁴C feedingto the tissue. Further proof for farnesol-2-¹⁴C incorporationinto ipomeamarone comes from the fact that the specific radioactivityof ipomeamarone semicarbazone was constant throughout the crystallizations.Fractionation of the label of farnesol-2-¹⁴C showed that radioactivitywas little distributed in the methanol-water fraction and wasmainly incorporated into ipomeamarone. Accordingly, it is notlikely that farnesol is incorporated into ipomeamarone afterits degradation to a small molecule(s) such as acetate. An additionalexperiment indicatedthat the incorporation of farnesol-2-¹⁴Cinto ipomeamarone markedly decreased under strict anaerobicconditions. This shows that some oxidative reactions are involvedin ipomeamarone biosynthesis from farnesol. ¹ This paper constitutes Part 91 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. (Received February 3, 1971; )     

The Oxidation of 14C-labelled Glucose by Chlorella vulgaris: 1. The Time Course of 14CO2 Production

Glucose, either uniformly labelled with¹⁴C, or specificallylabelled in the I, 2, or 6 position, was added to C. vulgaris.Radio-active carbon dioxide was produced initially ten timesfaster from glucose-I-¹⁴C than from glucose-6-¹⁴C. This differencewas found with carbohydrate-starved cultures, exponentiallygrowing cultures, and cultures assimilating ammonia or nitraterapidly. A similar difference was also found with C. pyrenoidosaand Ankistrodesmus. 37 per cent. of the ¹⁴C added as glucose-¹-¹⁴Cto exponentially growing cells was recovered as carbon dioxidebut generally the recovery was less than this. Only 5 per cent.of ¹⁴C added as glucose-6-¹⁴C was recovered as carbon dioxide.The specific activity of the carbon dioxide produced was considerablylower than that of the carbon in the added glucose.     

Cell Wall Metabolism in Developing Strawberry Fruits

Cell wall metabolism was studied in strawberry receptacles (Fragariaananassa, Duchesne) of known age in relation to petal fall (PF).Polysaccharide and protein composition, incorporation of [¹⁴C]glucoseand [¹⁴C]proline by excised tissue, and the fate of ¹⁴CO₂ fixedby young, attached fruits were followed in relation to celldivision, cell expansion, fine structure, and ethylene synthesis. Cell division continued for about 7 d after PF although vacuolationof cells was already beginning at PF and the subsequent cellexpansion was logarithmic. There was an associated logarithmicincrease in sugar content per cell and a decreasing rate ofethylene production per unit fresh weight. During cell expansion radioactivity from [¹⁴C]glucose was incorporatedinto fractions identified as starch and soluble polyuronideand into glucose and galactose residues in the cell wall. Radioactivityfrom [¹⁴C]proline was also incorporated into the cell wall,but only 10 per cent of this activity was found in hydroxyproline.Correspondingly wall protein contained a low proportion of hydroxyprolineresidues. The proportion of radioactivity from ¹⁴CO₂ fixed byfruitlets remained constant in most sugar residues in the cellwall. The proportion of radioactivity in galactose fell, indicatingturnover of these residues. Between 21 and 28 d after PF receptacles became red and softenedbut there was no change in the rate of ethylene production.Cell expansion continued for at least 28 d. Tubular proliferationof the tonoplast and hydration of middle lamella and wall matrixmaterial had begun 7–14 d after PF but became extremeduring ripening. Associated with the hydration of the wall,over 70 per cent of the polyuronide in the wall became freelysoluble, and arabinose and galactose residues lost from thewall appeared in soluble fractions. There was no increase intotal polysaccharide during ripening and incorporation of [¹⁴C]glucoseinto polysaccharides ceased, although protein increased andincorporation of [¹⁴C]proline into wall protein continued.     

The Use of C14-Proline by Growing Cell: Its Conversion to Protein and Hydroxyproline

C¹⁴-proline is readily absorbed by growing tissue cultures ofcarrot root phloem and of potato tuber in experiments carriedout under aseptic conditions. The C¹⁴-proline rapidly entersinto the protein of the tissue, appearing there in as shorta period as 15 minutes, and, thereafter, the amount incorporatedinto the protein bears a linear relation to time. Virtuallyall the C¹⁴ appears in the protein hydrolysate in the form ofproline and hydroxyproline. It is shown that the conversionfrom proline to hydroxyproline occurs after the C¹⁴-prolineis combined into the protein and that this conversion proceedsprogressively with time. The ratio of C¹⁴ as proline to C¹⁴as hydroxyproline declined progressively from a value of 4.0after 30 minutes of contact and seemed to become stabilizedeventually at 0.7. C¹⁴-hydroxyproline, which can be absorbedby the tissue, seems not to be incorporated into the proteinas such. The protein moiety which contains the C¹⁴-hydroxyprolinefrom C¹⁴-proline represents a stable protein which is not metabolizedand whose carbon does not ‘turn over’. This inertprotein seems to be characteristic of cells which are in rapiddivision under the influence of coconut milk or are synthesizingprotein in response to other stimuli such as the events at acut tissue surface. The protein in question seems to be presentmainly in the cytoplasm rather than in its paniculate inclusions.These results are compatible with earlier views which requirethat part of the protein in the cell ‘turns over’its carbon, whereas another part does not do so.     

Rhamnogalacturonan-II--a biologically active fragment

Rhamnogalacturonan-II inhibited the uptake of [¹⁴C]leucine and,consequently, the incorporation of [¹⁴C]leucine into acid-precipitableproteins by suspension-cultured tomato cells. Fractionationof rhamnogalacturonan-II showed that the lower molecular componentswere the most effective. KDO and apiose, both constituents ofrhamnogalacturonan-II, also inhibited [¹⁴C]leucine incorporationweakly, suggesting that these sugar residues may be an integralrequirement for the biological activity of rhamnogalacturonan-II.The incorporation of [¹⁴C]glutamate and [¹⁴C]histidine, andto a lesser extent [¹⁴C]proline and [¹⁴C]arginine, was alsoinhibited by rhamnogalacturonan-II; the incorporation of [¹⁴C]tyrosineand [¹⁴C]phenylalanine was little affected. This suggests thatrhamnogalacturonan-II exerts its effect by acting on certainmembrane transport systems. Key words: Rhamnogalacturonan-II, inhibition, protein synthesis, amino acid incorporation     

Storage Pools and Turnover Systems in Growing and Non-growing Cells: Experiments with 14C-Sucrose, 14C-Glutamine, and 14C-Asparagine

¹⁴C-labelled sucrose, glutamine, and asparagine have been suppliedto aseptically cultured carrot explants that either grew rapidlyby cell division or, by contrast, only slowly by cell expansion.The radioactive substrates were supplied in a brief ‘pulse’followed by a much longer period during which the tissues weresupplied with ¹²C-substrates. The passage of ¹⁴C through thevarious soluble compounds of the tissue and into the proteinwas followed. Alternatively, the ¹⁴C-labelled compound was suppliedthroughout the entire period of an experiment while the tissuealso received ¹²C-sucrose. The pulse-labelling experiments demonstrateturnover and the fate of the breakdown products, as well asthe emphasis placed on this kind of metabolism by cells at differentlevels of activity in their growth. The long-term labellingexperiments show the different ways in which carbon from varioussources may be used and how these pathways are affected by growth.The amount of ¹⁴C present in the various free (ethanol soluble)and combined (ethanol insoluble, acid-hydrolysable compounds—proteins)was determined, as well as the specific activity of the carbonin each compound. The fate of ¹⁴C supplied as sucrose had muchin common with ¹⁴C supplied as glutamine, with respect to theease with which it entered both the protein being synthesizedand the carbon dioxide evolved, but it was very different from¹⁴C supplied as asparagine. To interpret these data, compartmentsor pools of metabolites are postulated in the organized cell;exogenous ¹⁴C-sucrose and ¹⁴C-glutamine readily furnish carbonfor pools of amino-acids en route to protein, which are protectedfrom both the stored compounds and those which arise after proteinbreakdown. However, exogenous ¹⁴C-asparagine enters, is accumulated,and persists in the pool of stored compounds which also receivethe nitrogen-rich substances that arise from protein breakdown.The kinetic data and the specific activities of the carbon inits various forms require that protein breakdown and re-synthesisoccur concomitantly, that the stimulus to grow, exerted by coconutmilk, accentuates protein synthesis and also the pace of itsturnover, that some respired carbon dioxide arises from protein,and that this moiety of the respiration is increased by thecoconut-milk stimulus as it accentuates the pace of cyclicalturnover. In similar experiments with free cells from differentplants, the same general conclusions apply, but the rates ofturnover of protein are greater in free cells than in tissueexplants. Some specific differences, however, exist. Cells ofArachis, the only legume investigated, permit ¹⁴C-asparagineto contribute, like ¹⁴C-glutamine, to both protein synthesisand respired ¹⁴CO₂; it is not merely segregated in a storagepool. Thus, by virtue of their organization, plant cells maintainthe same substances simultaneously in distinct phases or compartments,where they play distinctive roles, without mingling. Geneticsendows each cell with the information that makes its biochemicalreactions feasible; the organization of the cells determineshow far the feasible becomes practised in cells in any givensituation.     

Fate of Immediate Methane Precursors in Low-Sulfate, Hot-Spring Algal-Bacterial Mats

The fates of acetate and carbon dioxide were examined in several experiments designed to indicate their relative contributions to methane production at various temperatures in two low-sulfate, hot-spring algal-bacterial mats. [2-¹⁴C]acetate was predominantly incorporated into cell material, although some ¹⁴CH₄ and ¹⁴CO₂ was produced. Acetate incorporation was reduced by dark incubation in short-term experiments and severely depressed by a 2-day preincubation in darkness. Autoradiograms showed that acetate was incorporated by long filaments resembling phototrophic microorganisms of the mat communities. [³H]acetate was not converted to C³H₄ in samples from Octopus Spring collected at the optimum temperature for methanogenesis. NaH¹⁴CO₃ was readily converted to ¹⁴CH₄ at temperatures at which methanogenesis was active in both mats. Comparisons of the specific activities of methane and carbon dioxide suggested that of the methane produced, 80 ± 6% in Octopus Spring and 71 ± 21% in Wiegert Channel were derived from carbon dioxide. Addition of acetate to 1 mM did not reduce the relative importance of carbon dioxide as a methane precursor in samples from Octopus Spring. Experiments with pure cultures of Methanobacterium thermoautotrophicum suggested that the measured ratio of specific activities might underestimate the true contribution of carbon dioxide in methanogenesis.     

Short-term Products of 14C-acetate Assimilation by Chlorella pyrenoidosa in the Light

The kinetics of ¹⁴C-2-acetate assimilation by Chlorella pyrenoidosain the light were examined. Under aerobic conditions the primaryproduct of acetate assimilation was succinic acid which, afterten seconds, contained over 60 per cent of the ¹⁴C incorporatedby the cells. The percentage of the total ¹⁴C in succinate fellwith time, while that in citrate and glutamate increased. After1800 sec over 60 per cent of ¹⁴C was present in two compounds,glutamic acid and an unknown compound (X). Glucose-6-phosphate,fructose-6-phosphate, phosphoglyceric acid and phosphoenolpyruvicacid became labelled after 60 sec but together never containedmore than one per cent of the total ¹⁴C incorporated. Underanaerobic conditions succinate was still the primary productof acetate assimilation, and the absence of carbon dioxide resultedin a decrease in ¹⁴C incorporation into compound X. The patternof acetate assimilation in acetate grown and acetate adaptedChlorella was very similar to that in photo-autotrophicallygrown Chlorella. In the presence of 10^–6M DCMU, succinicacid was the primary product of acetate assimilation, but therewas an early Incorporation of ¹⁴C into glutamate, aspartate,and malate. 4 x10^–3M MFA did not effect the early incorporationof ¹⁴C into succinic acid, but resulted in accumulation of ¹⁴Cin citrate and a decreased amount in glutamate and in compound X.     

Studies on the Cell Wall of Chlorella III. Incorporation of Photosynthetically Fixed Carbon into Cell Walls of Synchronously Growing Cells of Chlorella ellipsoidea

The carbon metabolism in cell walls of Chlorella ellipsoideawas studied by following ¹⁴C incorporation into cell wall constituentsin photosynthesizing, synchronously growing cells. The rateof incorporation was higher at an early growth phase of thecell cycle. The ¹⁴C was incorporated into both the major cellwall constituents, hemicellulose and ‘rigid wall’,and the radioactivity in the latter was distributed into itstwo components, glucosamine and amino acids. In pulse-chaseexperiments, the ¹⁴C fixed photosynthetically in the precedingcell cycle was rapidly transferred into the cell wall constituentsat the early growth phase of the ongoing cell cycle, and thereafterwas gradually released from the cell walls, although the totalamount of ¹⁴C in the cells remained constant. It was concludedthat the cell wall constituents are turned over during the growthphase of the algal cell cycle, and that the cell wall metabolismin the ongoing cell cycle is closely connected with the carbonmetabolism in the preceding cell cycle. (Received February 3, 1982; Accepted June 21, 1982)     

The Oxidation of 14C-labelled Glucose by Chlorella vulgaris: 2. The Fate of Radioactive Carbon

Most of the ¹⁴C added as glucose to carbohydrate-starved cellsof Chlorella Vulgaris can be recovered as alcohol-soluble compoundsor as polysaccharide. Only 5–I6 per cent., depending onthe position of ¹⁴C in the glucose supplied, is released ascarbon dioxide. Similar results were obtained with Chlorellapyrenoidosa and Ankistrodesmus. The labelled alcohol-solublecompounds in Chlorella vulgaris include amino-acids, particularlyglutamic acid, aspartic acid, and alanine, and, when glucose-I-¹⁴Cis metabolized, the amount of ¹⁴C recovered in these amino-acidsis about the same as that recovered as carbon dioxide. Degradationof the glucose incorporated into polysaccharide shown that theC₁ and C₆ atoms of glucose rapidly interchange when in the cells.The bearing of these results on attempts to estimate the relativeimportance of different pathways of glucose breakdown is discussed.     

BIOGENESIS OF ETHYLENE IN APPLE TISSUE: I. FORMATION OF ETHYLENE FROM GLUCOSE, ACETATE, PYRUVATE, AND ACETALDEHYDE IN APPLE TISSUE

1. With the aim of elucidating the path of carbon in the formationof ethylene in plants, studies were made on the incorporationof ¹⁴C into ethylene evolved from apple slices, using several¹⁴C- labeled compounds as substrates. The effects of inhibitorswere also investigated. 2. The formation of ethylene-¹⁴C from glucose-¹⁴C was inhibitedby fluoride, but unaffected by arsenite, thus suggesting thatglucose is converted to ethylene via pyruvate. 3. Acetate is converted to ethylene after cleavage of C-l andC-2. Only a small portion of the latter (C-2) enters the moleculeof ethylene, the former (C-l) is detected in carbon dioxide.On the other hand, 2, and 3-carbons of pyruvate are converted,without splitting, to ethylene. 4. On removal of air, the incorporation of ¹⁴C into ethylenefrom acetate-2-¹⁴C was depressed, while that from pyruvate-¹⁴Cwas unaffected. 5. Acetaldehyde-l,2-¹⁴C is converted to ethylene without conversioninto ethanol. 6. These results are interpreted to suggest the occurrence ofthe pathway in which pyruvate and acetaldehyde may serve asprecursors of ethylene. ¹ A part of this paper was read at the regular Meeting of KansaiBranch of the Agricultural Chemical Society of Japan in Kyoto,October, 1964, and at the Annual Meeting of the Japanese Societyof Plant Physiologists in Tokyo, April, 1965 and presented ina preliminary form elsewhere (10).     

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.     

Proline Metabolism in Tetrahymena1

SYNOPSIS. By the use of ¹⁴C-labeled substrates it has been shown in Tetrahymena that proline is rapidly and completely oxidized to carbon dioxide and glutamate (65–70%), plus small amounts of aspartate and alanine (20%), the remainder being incorporated into macromolecular cell components. In comparison, acetate, glucose and glutamate are oxidized to a lesser extent (55%, 37% and 16%, respectively). Glucose and acetate are extensively incorporated into cell components (53% and 36%, respectively), while glutamate remains in the medium (76%). Thus proline is a source of readily available energy.