Use of [3,4-14C]Glucose to Assess in vivo Competition for Phosphoenolpyruvate Between Phosphoenolpyruvate Carboxylase and Pyruvate Kinase in Developing Soybean Seeds

According to the conventional glycolytic sequence [3,4-¹⁴C]glucoseyields phosphoenolpyruvate (PEP) labeled in position C-1. Thisyields pyruvate through pyruvate kinase reaction also labeledin C-1. Subsequent metabolism of pyruvate to acetyl CoA releasesradioactive carbon dioxide. Alternatively PEP may be convertedto oxalacetate by PEP carboxylase and then into organic andamino acids which retain the label. The procedure adopted wasto trap carbon dioxide evolved and isolate organic acids producedafter feeding [3,4-¹⁴C]glucose to developing soybean cotyledons.Under conditions of 27?C and pH of 7.5 and 8.5 about 60% ofthe glycolytic carbon was processed by pyruvate kinase and 40%by PEP carboxylase. At lower temperature (15?C) 60% of the carbonwas directed through the PEP carboxylase reaction. This maybe caused by cold lability of pyruvate kinase which was demonstratedin in vitro assays. Low pH, down to 5.5, reduced organic acidproduction by inhibition of PEP carboxylase activity. Pyruvatekinase was not affected and carbon dioxide evolution remainedconstant at varying pH. PEP carboxyiase and pyruvate kinaseindependently feed their products into two separate metabolicpools. Possibly they should jointly be considered as final enzymesin the glycolytic pathway of plants. (Received April 3, 1982; Accepted June 12, 1982)     

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.     

The Reduction of ({+/-})-(2-14C) Abscisic Acid to the 1', 4'-Trans-Diol by Pea Seedlings and the Formation of 4'-Desoxy ABA as an Artefact

(±)(2-¹⁴C]ABA is metabolized by pea seedlings to phaseicand dihydrophaseic acids and conjugates and it is also reducedto the 1', 4'-trans-diol of ABA. This compound runs at the sameR_F as the putative metabolite identified as 4'-desoxy ABA byTietz et al (1979). The 1', m4'-/trans-diol is dehydrated duringgas/liquid chromatography to 4'-desoxy ABA. No labelled zoneon the autoradiogram of the plant extract could be attributedto 4'-desoxy ABA so it is presumably an artefact formed by thedehydration of the 1', 4'-trans-diol of ABA. This probably occursin the injection port of the G. L.C. Key words: Plsum sativum, A. B.A., Metabolism, Phaseic/dihydrophaseic acids     

The Resolution by HPLC of RS-[2-14C]Me 1', 4'-cis-Diol of Abscisic acid and the Metabolism of (-)-R- and (+)-S-Abscisic Acid

The R- and S-enantiomers of racemic [2-¹⁴C]Me 1', 4'-cis-diolof abscisic acid have been separated by high performance liquidchromatography on an optically-active Pirkle column. R-[2-¹⁴C]-and S-[2-¹⁴C]abscisic acids, formed from the Me 1', 4'-cis-diolby oxidation and alkyline hydrolysis were fed to tomato shootsand the extracts analysed by reversed phase high performanceliquid chromatography. R-[2-¹⁴C]abscisic acid formed mainlythe abscisic acid glucose ester (ABAGE), abscisic acid l'-glucoside(ABAGS) and an uncharacterized conjugate. Dihydrophaseic acid4'-B-D-glucoside, the major metabolite of RS-abscisic acid intomato shoots, was found to be derived virtually exclusivelyfrom the natural, S-abscisic acid. Phaseic acid and conjugatesof abscisic acid were also found as products of the naturallyoccurring enantiomer. The resolution method was used to measurethe relative proportions of R and S enantiomers in the freeacid liberated from conjugates formed from RS-[2-¹⁴C]ABA fedto shoots. The ratios show an excess of the R-enantiomer: 5.8:1, ABAGE; 29.4: 1, ABAGE; 8.3: 1 for an uncharacterized conjugateand 6.1: 1 for the residual free [2-¹⁴C]ABA. Key words: ABA, HPLC, Tomato     

Comparison of [14C]oryzalin Uptake in Root Segments of a Sensitive and a Resistant Species

Uptake of the dinitroaniline herbicide oryzalin (3,5-dinitro-N⁴,N⁴-dipropylsulfamlamide) and its effect on root growth werestudied using 5 mm corn (Zea mays L.) and pea (Pisum sativum)root apices. Pea root growth was much less susceptible to oryzalinthan corn root growth. Uptake studies showed that pea root apicesalso accumulated much less [¹⁴C]oryzalin and had a lower bindingaffinity for this herbicide. [¹⁴C]oryzalin was not metabolizedin root apices from either species. Thus, the differential susceptibilityto oryzalin in the case of corn versus pea can be explained,at least in part, by differences in oryzalin uptake and accumulationby roots. Oryzalin, dinitroaniline herbicides, Zea mays, Pisum sativum     

The Incorporation of 14CO2 into Green Peas in the Dark

Shelled green peas (ripening seeds of Pisum sativum var. Onward)were kept in the dark for 2 or 3 days in an atmosphere, eitherof air or 10 per cent carbon dioxide in air, containing ¹⁴CO₂.Samples were removed at intervals, the acids of the T.C.A.C.extracted, estimated, and their content of ¹⁴C measured. Incorporationof ¹⁴C was mainly into the carboxyl carbon atoms of the acidswhich, with the exception of citrate, rose in specific activityrapidly for the first 4–6 h and then levelled off androse slowly for the rest of the experiment. The final valuesattained, again with the exception of C-6 of citrate, were muchbelow that of the tissue carbon dioxide. The cause of this failureto equilibrate with tissue carbon dioxide is discussed. Twomain carboxylation reactions are proposed, one from pyruvate(or other three carbon acid) to malate and the other from -oxoglutarateto oxalsuccinate and so to C-6 of citrate. The value of thevelocity constant of the first of these reactions was shownto be markedly decreased by increase in tissue carbon dioxidewhile that for the second carboxylation was unaffected. ¹⁴Cmoved into soluble amino-acids rapidly at first and then moreslowly; protein received ¹⁴C at a high rate throughout the experimentsmainly by isotopic exchange reactions. Calculations were madeof the rate of movement of carbon in the T.C.A.C. which wouldhave been required to give the observed changes in specificactivity of the metabolites but no scheme tested fitted allthe results satisfactorily.     

ERRATA

Page 678, line 3, for [4-¹⁴C] read [I-¹⁴C] Page 678, line 4, for [I-¹⁴C] read [4-¹⁴C] Page 679, line 17, for C-I of malate read C-4 of malate Page 679, line 18, for C-4 of malate read C-I of malate     

The metabolism of L-[6-14C]ascorbic acid in detached grape leaves

Grape leaves (Vitis labrusca L.) that are removed from the positionopposite the flower cluster either 28 or 14 days before anthesiscleave L-ascorbic acid (AA) at the C4-C5 bond into a C₄ and,presumably, a C₂ fragment. Leaves taken from this position 14days after anthesis fail to cleave AA. The C₄ fragment is utilizedfor L(+)-tartaric acid (TA) biosynthesis while the C₂ fragmentis recycled into hexose and products of the hexose metabolism.When [6-₁₄C]AA is the source of the label, the sucrose-derivedglucose from labeled leaves has a distribution of ₁₄C in thecarbon skeleton as follows: Cl, 35%; C2, 14%; C3, 4%; C(4+5),13% and C6, 34%. The effect of inhibitors of the glycolate pathwayon [6-^l4C]AA metabolism is examined. ¹This work was supported by Grant No. GM-22427 from the NationalInstitute of General Medical Science, National Institute ofHealth, United States Public Health Service, Bethesda, Maryland.This is Scientific paper No. 5305, Project 0266, College ofAgricultural Research Center, Washington State University, Pullman,WA 99164, U.S.A. ²Present address: The Radioisotope Research Center, Kyoto University,Kyoto 606, Japan. (Received August 29, 1979; )     

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.     

Long-term Metabolism of [14C]Sugars in Stored Potatoes

[¹⁴C]Sucrose, [¹⁴C]glucose and [¹⁴C]fructose were introducedinto potato tubers held at 10 °C and the redistributionof label chased over a 65 d period in storage. Respiratory losseswere identical in all treatments, as was the partitioning of¹⁴C between soluble and insoluble forms. Sucrose was the predominantlabelled sugar in the tubers after 20 h, regardless of the original[¹⁴C]sugar introduced, and was loaded and distributed throughoutthe tubers by the internal phloem system. After 20 h the proportionsof labelled sugars bore no relationship to those of the unlabelledendogenous sugars. However, with time the percentage of ¹⁴Cin sucrose fell while that in glucose increased and by 65 dthe proportions of the labelled sugars more closely resembledthe endogenous pools. Fructose represented a consistently lowproportion of both the labelled and unlabelled sugars. By 21d a considerable proportion of the soluble ¹⁴C had been convertedto starch (approx. 25% of the total tuber 14C), this value remainingrelatively constant for the remainder of the storage period.Sprouts which formed on the tubers contained up to 6% of thetotal tuber ¹⁴C but less than 0.2% of the tuber dry matter.It is suggested that the bulk of the translocated [¹⁴C]sucroseentered the symplast and exchanged slowly with the bulk of thesugars in the storage cell vacuoles. [¹⁴C]sugars, phloem loading, starch, potato tuber, Solunum tuberosum, cold storage     

Hormones in Plants Bearing Nitrogen-fixing Root Nodules: Metabolism of [8-14C]Zeatin in Root Nodules of Alnus glutinosa (L.) Gaertn

The metabolism of [8-¹⁴C]zeatin, supplied via micropipettesover a 24 h period to root nodules of Alnus gliutinosa (L.)Gaertn., was investigated. The major metabolites were tentativelyidentified by means of chromatographic, chemical, and enzymictreatments as adenine, adenosine, trans-zeatin riboside, dihydrozeatin,trans-zeatin-O-ß-D-glucoside, and the O-ß-D-glucosideof dihydrozeatin. In addition, a prominent water-soluble peakof radioactivity was present. This did not appear to be a ribosidebut was biologically active in the soybean callus test. The number and nature of the metabolites formed in the noduleswas similar in both dormant and non-dormant plants.     

The mechanism supplying acetyl-CoA for terpene biosynthesis in sweet potato with black rot: Incorporation of acetate-2-14C, pyruvate-3-14C and citrate-2,4-14C into ipomeamarone

Silica gel thin layer chromatography showed that acetate-2-¹⁴C,pyruvate-3-¹⁴C and citrate-2,4-¹⁴C were incorporated into ipomeamaronein sweet potato root tissues infected by Ceratocystis fimbriata.Rates of incorporation of ¹⁴C, from these 3 substances, intothe CHCl³-CH₃OH-soluble lipid fraction and ipomeamarone wereof the followingder: acetate > pyruvate > citrate ¹This paper constitutes Part 82 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury (Received December 11, 1969; )     

Metabolic patterns of 14C incorporation by selected vascular plants following field incubations with acetate-2-14G in two plant successional stages in Glacier Bay, Alaska

Metabolic patterns of some vascular plants (Dryas sp., Vacciniumsp., Salix sp., Alnus sp., Epilobium sp.), occurring in successionalhabitats, following acetate-2-¹⁴C incubations in the field weredemonstrated for the first time. Relative radioactivity withinthe alcoholic soluble fraction of each species reflects itsdistribution in successional communities. A high level of ¹⁴C-sugarswas present in the plants of the pioneer community; on the otherhand a high level of ¹⁴C-organic acids was present in the plantsof the forest community. Three patterns, based on the relativeactivities of the sugar- and organic acid-pools were noted whichcorrespond to the range and the frequency of occurrence of eachspecies in the successional stages. Only two types of ¹⁴C-aminoacid levels were noted corresponding to the range of distribution.Plants having less than 10% relative radioactivity in aminoacid-pools had a limited range of distribution and reside inonly one habitat; plants having more than 10% radioactivityshowed wider ranges of distribution occurring in at least twohabitats. ¹ Contribution 292 of the Institute of Polar Studies, The OhioState University, Columbus, Ohio 43210. (Received April 16, 1975; )     

Uptake, transport and stability of [2-14C] abscisic acid in cucumber following foliar application

The uptake, transport and stability of ABA after foliar applicationto cucumber plants was studied using [2-¹⁴C] ABA. ABA uptakethrough uninjured epidermis of cucumber leaves appeared to bevery slow compared with the uptake through abraded leaves. Transportof [2-¹⁴C] ABA in the same system was also found to be veryslow. No metabolic changes in the absorbed [2-¹⁴C] ABA weredetected 24 hr after its application as checked by TLC and GLCtechniques. These results are compared and discussed in relationto previous reports. ¹The Irene and David Schwartz Professor of Plant Genetics (Received April 26, 1976; )     

Technique for Simultaneous Determination of [35S]Sulfide and [14C]Carbon Dioxide in Anaerobic Aqueous Samples

A technique for the simultaneous determination of [³⁵S]sulfide and [¹⁴C]carbon dioxide produced in anaerobic aqueous samples dual-labeled with [³⁵S]sulfate and a ¹⁴C-organic substrate is described. The method involves the passive distillation of sulfide and carbon dioxide from an acidified water sample and their subsequent separation by selective chemical absorption. The recovery of sulfide was 93% for amounts ranging from 0.35 to 50 μmol; recovery of carbon dioxide was 99% in amounts up to 20 μmol. Within these delineated ranges of total sulfide and carbon dioxide, 1 nmol of [³⁵S]sulfide and 7.5 nmol of [¹⁴C]carbon dioxide were separated and quantified. Correction factors were formulated for low levels of radioisotopic cross-contamination by sulfide, carbon dioxide, and volatile organic acids. The overall standard error of the method was ±4% for sulfide and ±6% for carbon dioxide.     

Metabolism of [14C]Indole-3-Acetic Acid by Coleoptiles of Zea mays L.

Reverse-phase high-performance liquid chromatography was usedto analyse [¹⁴C]-labelled metabolites of indole-3-acetic acid(IAA) in coleoptile segments of Zeo mays seedlings. After incubationfor 2 h in 10^–2 mol m^–3 [2-¹⁴C]IAA, methanolic extractsof coleoptiles contained between six and ten radioactive compounds,one of which co-chromatographed with IAA. The metabolic productsin coleoptile extracts appeared to be similar to those in rootextracts, with an oxindole-3-acetic-acid-like component as theprincipal metabolite, but the rate of metabolism was slowerin coleoptile than in root segments. Decarboxylation did notappear to play a major role in the metabolism of exogenous IAAduring the short incubation periods. Moreover, external IAAconcentration had little effect on the pattern of metabolism.Coleoptile segments were also supplied with [¹⁴C]IAA from agardonor blocks placed at the apical ends, and agar receiver blockswere placed at the basal ends. After incubation for 4 h, theidentity of the single radioactive compound in the receiverblocks was shown to be IAA by both reverse-phase high-performanceliquid chromatography and gas chromatography-mass spectrometrytechniques. Key words: Zea mays, Coleoptile, High-performance liquid chromatography, Indole-3-acetic acid     

[14C]-Glucose Metabolism during Shoot Bud Development in Cultured Cotyledon Explants of Pinus radiata

Excised cotyledons of Pinus radiata D. Don cultured under shoot-forming(plus benzyladenine) and non shoot-forming (minus benzyladenine)conditions for 10 and 21 days were fed U-[¹⁴C]-glucose for 3h in the light followed by a 3 h chase period. The labellingof individual metabolites as well as ¹⁴C incorporation intoprotein was assessed. It was found that the general metabolicpatterns were qualitatively the same in shoot-forming and nonshoot-forming conditions, however, metabolism leading to respirationas well as to the synthesis of some amino acids and proteinsynthesis was enhanced in the shoot-forming cultures. (Received February 16, 1987; Accepted July 8, 1987)     

The Partitioning of Photosynthetically Assimilated 14C into Amino Acids in Wheat 1. Partitioning During Transport to the Grain

When ¹⁴CO₂ was fed to flag leaf laminae at 20 d post-anthesis,the transport organs between the leaf and the grains containedappreciable ¹⁴C in glutamine, glutamate, serine, alanine, threonineand glycine. Smaller amounts of ¹⁴C were present in gamma-aminobutyricacid (GABA), aspartate and cysteine. Other amino acids whichwere labelled in the source leaf were not labelled in the transportorgans. The export of labelled glutamine, serine, glycine andthreonine from the source leaf was favoured in comparison tothe other amino acids mentioned. Threonine accumulated, andwas subsequently metabolised, in the rachis. [¹⁴C]GABA alsoaccumulated in the rachis. In the grains, the relative amountof soluble [¹⁴C]alanine increased with chase time. This wasprobably due to de novo synthesis and reflected the specialrole of alanine in grain nitrogen metabolism. Wheat, Triticum aestivum, ¹⁴CO₂, amino acids, transport, carbon metabolism     

The Translocation of 14C-Photoassimilate from Normal and Mutant Leaves to the Pods of Pisum sativum L.

In experiments using radioactive carbon dioxide (¹⁴CO₂) a comparisonwas made of the ¹⁴C-photoassimilate translocation potentialsof two normal leaved (genotype AfAfTlTl) and two mutant formsof Pisum sativum (pea). A ¹⁴CO₂ administration method is describedthat permitted ¹⁴C-translocation studies to be conducted underfield conditions. One of the mutants available produced tendrils in place of leaves(afafTlTl). The other mutant studied was without tendrils buthad a much branched petiole with numerous relatively minuteleaflets (afaftltl). These mutants and the normal-leaved cultivarswith which they were compared were not isogenic lines. Lengthybackcrossing would be required before full assessment couldbe made of the possible agronomic value of such mutations. An interim evaluation of these mutants was based on ¹⁴C-distributionassays that were conducted 48 h after feeding ¹⁴CO₂, to specifiedleaves. The indication was that in translocation terms the leafand pod had a well defined respective source and sink relationshipthat was independent of leaf morphology. In each case the podswhich constituted the major ¹⁴C sinks depended on which leafhad been fed ¹⁴CO₂. With regard to sink specific activity asdefined by the quantity of ¹⁴C incorporated per unit dry weightof pod, the mutants were not significantly different from normal. The implication of these findings was that fundamental changesin pea leaf morphology could be made genetically without a markedeffect on the photoassimilate export potential of the leaf.