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; )     

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; )     

Fatty acid biosynthesis from acetate and CO2 in the developing soybean cotyledon

Developing soybean cotyledons rapidly incorporated acetate intofatty acids and water soluble constituents. Oleic acid was thefirst fatty acid to be detected with ¹⁴C and the ¹⁴C distributionpattern with time was consistent with its being the precursorof linoleic and linolenic acids. Palmitic or stearic acid didnot appear to be the precursor of oleic acid but appeared tobeformed parallel to it. The cotyledons did fix ¹⁴CO₂ by eitherdark or light fixation reactions but little ¹⁴C was incorporatedinto lipids. ¹Presented in part at the Midwest Section of the American Societyof Plant Physiologist Meeting in Columbia, Missouri, June 1970 (Received November 27, 1970; )     

Studies on chlorogenic acid biosynthesis in sweet potato root tissue using trans-cinnamic acid-2-14C and quinic acid-G-3H

When either trans-cinnamic acid-2-¹⁴C or quinic acid-G-³H wasadministered to sweet potato root discs, each compound was incorporatedinto chlorogenic acid. Hydrolysis analysis revealed that trans-cinnamicacid-2-¹⁴C and quinic acid-G-³H were selectively incorporatedinto the aromatic and non-aromatic moieties of chlorogenic acid,respectively. Quinic acid-G-³H was considered a more efficient precursor thantrans-cinnamic acid-2-¹⁴C, based on data of dilution values,incorporation percents and pool sizes in the tissue. No conjugatesof trans-cinnamic acid and quinic acid were detected in discsadministered trans-cinnamic acid-2-¹⁴C or quinic acid-G-³H.From these experimental results, a possible biosynthetic pathwayfor chlorogenic acid has been proposed. ¹ This paper constitutes Part 98 of the Phytopathological Chemistryof Sweet Potato with Black Rot or Injury. (Received November 2, 1971; )     

Biosynthesis of Acyl Moieties of Capsaicin and its Analogues from Valine and Leucine in Capsicum Fruits

Biosynthetic pathways of acyl moieties of capsaicinoid in intactCapsicum fruits and spheroplasts prepared from placentas ofCapsicum fruits were examined using a radioisotopic technique.In intact Capsicum fruits, L-[U-¹⁴C] valine was incorporatedinto capsaicin and dihydrocapsaicin, the acyl constituents ofwhich are even-number branched chain fatty acids, while L-[U-¹⁴C]leucine was incorporated into nordihydrocapsaicin and homodihydrocapsaicin,which have odd-number branched chain facty acids as the acylmoieties. The intermediates of the odd- and even-number branchedchain fatty acids were identified with GLC/GPC after the spheroplastshad been incubated with L-[U-¹⁴C] valine or L-[U-¹⁴C] leucine.After incubation with L-[U-¹⁴C] valine, isobutyric acid and8-methyl nonanoic acid were detected, while isopentanoic acidand 9-methyl decanoic acid were found after incubation withL-[U-¹⁴C] leucine. The involvement of a-ketoisovalerate or a-ketoisocaproatein the biosynthesis of acyl moieties of capsaicinoid was alsodemonstrated in vitro using cell-free extracts of the placentasof Capsicum fruits. These findings suggest that the acyl moietiesof individual capsaicinoids in Capsicum fruits are synthesizedby pathways similar to those proposed for adipose tissue andbacteria. ¹Formation and Metabolism of Pungent Principle of Capsicum Fruits.Part IX. (Received September 2, 1980; Accepted November 17, 1980)     

FORMATION OF LIGNIN IN TISSUE CULTURE OF PINUS STROBUS

The formation of shikimic acid and lignin from glucose in thecambium tissue was investigated. Glucose-1-¹⁴C, shikimic acid-G-¹⁴C, sodium acetate-1-¹⁴C and sodium acetate-2-¹⁴C were administeredto the tissue culture of strob pine. Glucose was well incorporatedinto shikimic acid, but acetic acid was less effective. Shikimicacid was very efficient as a precursor of aromatic nucleus andglucose was also converted efficiently to lignin. The extentof incorporation of acetic acid, however, was considerably low.A possibility was discussed that in the cultured tissue ligninand its precursor were synthesized from glucose via the shikimicacid pathway. (Received May 14, 1960; )     

Metabolism of Pyrimidines in Protoplasts from Cultured Catharanthus roseus Cells

The metabolism of [2-¹⁴C]thymine, [2-¹⁴C]thymidine, [2-¹⁴C]uraciland [¹⁴C]uridine was investigated in protoplasts obtained fromsuspension cultures of Catharanthus roseus. Most of the exogenouslysupplied thymine, thymidine and uracil was degraded, and salvageof these pyrimidines accounted for 5–36 per cent of thetotal amount of ¹⁴C-labelled precursors which was metabolized.However, more than 80 per cent of the labelled uridine was utilizedfor the biosynthesis of nucleotides and nucleic acids, and therest was degraded. In contrast to the results from protoplastsof sugar cane cells in suspension culture, the data indicatethat protoplasts possess a pathway for the degradation of pyrimidines,and that the overall metabolism of these pyrimidines in protoplastsis very similar to the metabolism in the intact cells. Catharanthus roseus, madagascar periwinkle, protoplasts, pyrimidine metabolism     

Studies on the Growth in Culture of Plant Cells: XV. UPTAKE AND UTILIZATION OF URIDINE DURING THE GROWTH OF ACER PSEUDOPLATANUS L. CELLS IN SUSPENSION CULTURE

[2-¹⁴C]-uridine is rapidly taken up by sycamore cells in suspensionculture. A proportion of the radioactivity enters RNA withoutmeasurable delay, whilst the remainder equilibrates with a largepool of phosphorylated compounds, the major radioactive componentof which is 5'-UMP. Both the uracil and cytosine residues ofRNA receive label from [¹⁴C]-uridine and, when the cells aresupplied with high concentrations of uridine, these bases arederived almost exclusively from the nucleoside. [¹⁴C]-uridine is incorporated into RNA at all stages of thegrowth cycle of batch cultures; its continuing incorporation,when the total RNA content of the cells is rapidly decreasing,indicates a high rate of turnover of the total RNA. Long-termlabelling experiments also indicate turnover of RNA during thephase of active cell division and suggest that a large proportionof the degradation products are not re-utilized for RNA synthesis. Sycamore cells degrade [2-¹⁴C]-uridine with release of ¹⁴CO₂.The proportion degraded increases from 25 per cent at an externaluridine concentration of 10^–6M to 75 per cent at 10^–3M. Despite this, nucleic acids are the only macromolecules thatreceive a significant amount of radioactivity from [2-¹⁴]C-uridine.     

Germination of Phaseolus vulgaris III. The role of nucleic acid and protein synthesis in the initiation of axis elongation

Excised embryonic axes of Phaseolus vulgaris L. (var. WhiteMarrowfat) begin cell elongation after approximately 4 hr ofincubation at 26°C. The incorporation of ³²P into nucleicacids and phenylalanine-l-¹⁴C into protein markedly increasesduring the 4th hr of incubation, prior to initiation of cellelongation. CH, which inhibits incorporation of phenylalanine-l-¹⁴C intoprotein by 93% during the 2nd hr after its addition, completelyprevents the initiation of axis elongation if added up to 2hr after the beginning of imbibition. Actinomycin D reducesthe fresh weight increase of the axes, and inhibits both ³²Pincorporation into nucleic acids and phenylalanine-l-¹⁴C incorporationinto protein. 5-FU inhibits ³²P incorporation into nucleic acidsbut not phenylalanine-l-¹⁴C incorporation into protein or thefresh weight increase of the axes. MAK column chromatography indicates that actinomycin D inhibitsthe synthesis of all types of nucleic acids to about the sameextent, while 5-FU almost completely inhibits the accumulationof ³²P in ribosomal RNA with lesser but significant inhibitoryeffects on accumulation of ³²P in tRNA. The results suggest an absolute requirement for protein synthesisprior to initiation of cell elongation and at least a partialrequirement for synthesis of nucleic acid species other thanribosomal RNA, tRNA and DNA. The kinetic data suggest that theaxes develop a greatly increased capacity for nucleic acid andprotein synthesis prior to initiation of axis elongation. ¹This research was supported by NSF grant GB 4145 and a grantfrom the U. S. Forest Service. (Received December 16, 1968; )     

Cytokinins in Germinating Seeds of Phaseolus vulgaris L. III. Transport and Metabolism of 8[14C]t-Zeatin Applied to the Radicle

The application of 8[¹⁴C]t-zeatin to the cotyledons of germinatingbean seeds demonstrated that cytokinins are not readily exportedfrom the cotyledons to the embryonic axis during the early stagesof this process. In the cotyledons the applied zeatin is metabolizedextensively to metabolites which are polar and which occur atR_F 0·2–0·5 on paper chromatograms. Thesemetabolites are stable and are not readily exported from thecotyledons. In contrast the metabolites found at R_F 0–0·2are more readily exported. When exported to the radicles andplumules a large proportion of the translocated metaboliteswere converted to compounds which on paper co-chromatographedwith zeatin. This seems to suggest that the embryonic axis hasthe capacity to synthesize cytokinins and that some of the metabolitesformed during its catabolism can also be used for its synthesis. Phaseolus vulgaris, bean, germination, cytokinins, transport, cotyledons     

Effect of indoleacetic acid on the penetration of leucine and glucose through epidermal tissue of onion bulb

The penetration of leucine-(U)-¹⁴C and glucose-(U)-¹⁴C throughthe bulb epidermal tissue of Allium cepa was examined in thepresence of indoleacetic acid (IAA). Not only the uptake ofleucine-¹⁴C and glucose-¹⁴C in epidermal tissue but also theirtranscellular penetration were accelerated by IAA treatment.N-Ethylmaleimide (NEM) inhibited their uptake and transcellularpenetration, and the inhibitory effect was relieved by additionalIAA. In the presence of IAA, leucine-¹⁴C and glucose-¹⁴C weremore penetrable by adaxial than abaxial application, but inthe absence almost no difference due to application side wasobserved. IAA appears to promote permeability of the epidermaltissue only to substances applied adaxially. N,N'-Dicyclohexylcarbodiimide(DCCD) showed a little inhibitory effect on the IAA-inducedpromotion of the uptake and penetration of leucine-¹⁴C appliedadaxially. Leucine-¹⁴C and glucose-¹⁴C penetrated more easilythrough killed than fresh tissue, with little difference betweenabaxial and adaxial applications. ¹ Present address: Department of Biology, Faculty of Science,Kochi University, Kochi 780, Japan. ² Present address: Department of Medical Zoology, Medical School,Mie University, Tsu 514, Japan. (Received October 13, 1977; )     

Metabolism of C1 unit precursors in Neurospora: Effects of media supplements on labelling of nucleic acids and protein

Mycclia of Neurospora crassa wild type (FE SC no. 853), harvestedduring the exponential phase of growth on defined minimal mediaincorporated glycine-2-¹⁴C, serine-3-¹⁴C and formate-¹⁴C intoproteins, DNA and RNA. Supplementing the growth medium with1 mM glycine increased the flow of glycine and formate carboninto these products. In contrast, this supplement decreasedthe incorporation of serine-¹⁴C. When such cultures were preincubatedfor 30 min with adenine, formaldehyde, formate or L-methionine,labelling of the nucleic acids and protein fractions by glycine-2-¹⁴Cwas altered. It is concluded that glycine increases the turnoverof C₁ units in Neurospora, resulting in greater contributionsof the C-2 in nucleic acid and protein synthesis. (Received May 14, 1977; )     

Salinity Stress and the Content of Proline in Roots of Pisum sativum and Tamarix tetragyna

Amino acid composition of the free amino acid pool and the TCA-insolubleprotein fraction were investigated in root tips of pea and Tamarixtetragyna plants grown at various levels of NaCl salinity. Salinitystress induced an increase of proline content, mainly in thefree amino acid pool in both plants, and of proline or hydroxyprolinecontent in the protein. Externally-supplied proline was absorbedand incorporated into protein, by pea roots, more effectivelythan by Tamarix roots. Salinity stress, apparently, stimulatedthe metabolism of externally-supplied labelled proline. Pearoots have a very large pool of free glutamic acid; however,70 per cent of the ¹⁴C from externally-supplied ¹⁴C-U-glutamicacid was released as CO₂. Very small amounts of it were incorporatedinto protein. No measurable amount of radioactivity could bedetected in any one of the individual amino acids, either ofprotein hydrolysate or the free amino acid pool. Proline very effectively counteracted the inhibitory effectof NaCl on pea seed germination and root growth. A similar effectbut to a lesser degree was achieved with phenylalanine and asparticacid. The feasibility of proline being a cytoplasmic osmoticumis discussed.     

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.     

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.     

Biosynthesis of Resin Terpenes in Leaves and Glandular Hairs of Newcastelia viscida

Label from ¹⁴CO₂ and [2-¹⁴C]mevalonic acid was incorporatedinto resin components of Newcastelia viscida. About 25% of thelabel from [2-¹⁴C]MVA was incorporated into resin componentsafter 4 h. Approximately 80% of the label in the resin was ina TLC band coinciding with the major terpenoid components, with60% associated with the major tricyclic diterpene, a pimaradienewhich contributes 15% to the leaf resin dry weight. Experimentswith detached trichomes showed about 0?01% incorporation oflabel from [2-¹⁴C]MVA into pimaradiene, with most of the [abelbeing associated with ß-sitosterol. It is proposedthat the glandular hairs are the sites of terpene resin synthesis,as well as secretion, in this species.     

Transport and Metabolism of Indol-3yl-(Acetic Acid-2-14C) in Pea Roots

¹⁴C from indol-3-yl-(acetic acid-2-¹⁴C) (IAA-¹⁴C) was transportedin a weak but definitely polar manner through segments of youngand matured regions of pea roots. Greater quantities of ¹⁴C-labelledmaterial moved acropetally than basipetally. Up to 70 per centof radioactivity originally present in donor agar blocks wastaken up by the root segments, but only approximately 2 to 3per cent of this emerged into the receiver agar blocks. Anydifferences in uptake, transport, or binding of auxin were veryslight in the three regions of root studied. The IAA-¹⁴C wasmetabolized during passage through the root segments, yieldingtwo principal radioactive products. The identities of thesewere not determined, but they appeared to have auxin activityand may be formed spontaneously, but more slowly, in solutionsof IAA-¹⁴C. IAA-¹⁴C was transported into receiver blocks morereadily than its radioactive derivatives.     

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.     

Biosynthesis of Purine Alkaloids in Camellia Plants

The metabolism of [8-¹⁴C]adenine and [8-¹⁴C]hypoxanthine infour species of Camellia plants was investigated in relationto the synthesis of purine alkaloids, caffeine and theobromine.Young leaves of C. sinensis had the ability to synthesize caffeine,but in C. irrawadiensis, these labelled precursors were incorporatedinto theobromine, not caffeine. No synthesis of purine alkaloidscould be detected in C. japonica and C. sasanqua leaves. Conventional"salvage" and degradation pathways of purines were present inall species of Camellia plants examined. ¹ Present address: Research Center, Mitsubishi Chemical IndustriesLtd., 1000 Kamisida-cho, Midori-ku, Yokohama, 227 Japan. (Received September 29, 1986; Accepted January 22, 1987)     

Metabolism of arginine by aging and 7 day old pumpkin seedlings

The metabolism of arginine by etiolated pumpkin (Cucurbita moschata) seedlings was studied over various time and age intervals by injecting arginine-U-¹⁴C into the cotyledons. At most, 25% of the ¹⁴C was transported from the cotyledon to the axis tissue and the amount of this transport decreased with increasing age of the seedlings. The cotyledons of 25 day old plants contained 60% of the administered ¹⁴C as unmetabolized arginine. Little ¹⁴C was in sugars and it appeared that arginine was the primary translocation product. Time course studies showed that arginine was extensively metabolized and the labeling patterns suggest that different pathways were in operation in the axis and cotyledons. The amount of arginine incorporated into cotyledonary protein show that synthesis and turnover were occurring at rapid rate. Only 25% of the label incorporated into protein by 1.5 hr remained after 96 hr. The label in protein was stable in the axis tissue. By 96 hr 50% of the administered label occurred as ¹⁴CO₂ and it appeared that arginine was metabolized, through glutamate, by the citrio acid cycle in the cotyledons. The experiments showed that an extensive conversion of arginine carbon into other amino acids did not occur.