Labeled indole-macromolecular conjugates from growing stems supplied with labeled indoleacetic Acid : I. Fractionation

Pea (Pisum sativum var. Alaska) and bean (Phaseolus vulgaris var. Red Kidney) stem sections treated with indoleacetic acid-1-¹⁴C, indoleacetic acid-2-¹⁴C, and indoleacetic acid-5-³H were homogenized, extracted with phenol, and the water-soluble, ethanol-insoluble material subjected to further fractionation. Following an 18-hour incubation period in indoleacetic acid-1-¹⁴C, most of the label was found as nonindole-¹⁴C in high molecular weight polysaccharide, as phenol extraction is specific for both RNA and polysaccharides. With indoleacetic acid-2-¹⁴C and -5-³H, and to a lesser extent with indoleacetic acid-1-¹⁴C, radioactive indoles were obtained by hydrolysis from a heterogeneous fraction between about 500 and 30,000 molecular weight, possibly polysaccharide in nature. Indoleacetic acid accounted for 8% and indole aldehyde accounted for 21% of the total radioactivity in the extract.     

Participation of C6C1 unit in the biosynthesis of ephedrine in Ephedra

Feeding of benzoic acid-[7-¹⁴C], benzaldehyde-[7-¹⁴C] and cinnamic acid-[3-¹⁴C] to Ephedra distachya resulted in efficient incorporations of ¹⁴C into the α-carbon atom of the side chain of l-ephedrine. Thus ephedrine was shown to be biosynthesized by the condensation of a C₆C₁ portion which is derived from phenylalanine via cinnamate and an unidentified C₂-N fragment.     

Biosynthesis of p-hydroxybenzoic acid in poplar lignin

Biosynthetic pathways to p-hydroxybenzoic acid in polar lignin were examined by tracer experiments. High incorporation of radioactivity to the acid was observed when shikimic acid-[1-¹⁴C], phenylalanine-[3-¹⁴C], trans-cinnamic acid-[3-¹⁴C], p-coumaric acid-[3-¹⁴C] and p-hydroxybenzoic acid-[COOH-¹⁴C] were administered, while incorporation was low from shikimic acid-[COOH-¹⁴C], phenylalanine-[1-¹⁴C], phenylalanine-[2-¹⁴C], tyrosine-[3-¹⁴C], benzoic acid-[COOH-¹⁴C], sodium acetate-[1-¹⁴C] and d-glucose-[U-¹⁴C]. Thus p-hydroxybenzoic acid in poplar lignin is formed mainly via the pathway: shikimic acid → phenylalanine → trans-cinnamic acid → p-coumaric acid → p-hydroxybenzoic acid.     

The stereochemistry of biosynthesis of decaprenoxanthin in a cell-free system

Intact cells of Flavobacterium dehydrogenans grown on glucose or acetate did not incorporate mevalonic acid-[¹⁴C]. After treatment with lysozyme the protoplasts were lysed by sonication in a dilute medium containing mevalonic acid-[¹⁴C] and the cell-free system produced incorporated label into uncyclized C₄₀, monocyclic C₄₅ and bicyclic C₅₀ carotenoids of which decaprenoxanthin was the most abundant.With mevalonate-[2-¹⁴C,4R-4-³H₁] the ¹⁴C:³H ratios of the carotenoids showed that the hydrogen atoms at C-2 and C-6 of the ring and that at C-3 of the 1-hydroxy, 2-methyl but-2-ene-4-yl residues of decaprenoxanthin were derived from the 4-pro-R hydrogen atom of mevalonic acid.Mevalonate-[2-¹⁴C,2R-2-³H₁] and mevalonate-[2-¹⁴C,2S-2-³H₁] gave ratios which showed that the C-4 hydrogen atoms of decaprenoxanthin were derived from the 2-pro-S hydrogen atom of mevalonic acid.     

Biosynthesis of the tigloyl esters in Datura: The role of 2-methylbutyric acid

Datura innoxia plants were wick fed with (±)-2-methylbutyric acid-[1-¹⁴C] and harvested after 7 days. The root alkaloids 3α,6β-ditigloyloxytropane and 3α,6β-ditigloyloxytropan-7β-ol were isolated and degraded. In each case the radioactivity was located in the ester carbonyl group indicating that this acid is an intermediate in the biosynthesis of tiglic acid from l-isoleucine. On the other hand, (±)-2-hydroxy-2-methylbutyric acid-[1-¹⁴C], which was fed to hydroponic cultures of Datura innoxia alongside isoleucine[U-¹⁴C] positive control plants, is not an intermediate.     

Kinetic study of possible intermediates in the biosynthesis of chlorogenic acid in Cestrum poeppigii

p-Coumaric and 3-O-p-coumarylquinic acid seem to be important precursors of chlorogenic acid in the leaves of Cestrum poeppigii. 3-O-Cinnamylquinic acid, which has a very small metabolic activity, is of little importance in this respect. The kinetics of incorporation of radioactivity from t-cinnamic acid-3-[¹⁴C] into p-coumaric, 3-O-p-coumarylquinic, chlorogenic and 3-O-cinnamylquinic acid showed that the biosynthetic rates for these products decrease in the order shown. For p-coumaric acid, which has a markedly high metabolic activity, a turnover rate of 28 μg/hr and per gram fresh plant leaf, was calculated. Some trapping experiments with caffeic acid, and the acids mentioned above and using either t-cinnamic acid-3-[¹⁴C] or p-coumaric acid-2-[¹⁴C] as precursor, are discussed. A HPLC method for the rapid determination of phenolic acids in plant extracts, is described.     

Biosynthesis of 3α,6β-ditigloyloxytropane and 3α,6β-ditigloyloxytropan-7β-ol in Datura

Datura meteloides; plants were fed with tiglic acid-[-¹⁴C] via the roots and after 2 days the percentage incorporation into the alkaloids 3α-tigloyloxytropane, 3α,6β-ditigloyloxytropane, meteloidine and 3α,6β-ditigloyloxytropan-7β-ol were 15·2, 1·82, 2·2 and 1·8 respectively. 3α,6β-Ditigloyloxytropane was partially hydrolysed to 6β-hydroxy-3α-tigloyloxytropane which contained 58·1% of the radioactivity of the original base, whereas 3α,6β-ditigloyloxytropan-7β-ol gave meteloidine containing only 9·2% of the original activity. The results suggest that the di- and tri-hydroxytropanes may be formed by different routes.     

Elongation of acyl-CoAs by microsomes from etiolated leek seedlings

Long chain fatty acid synthesis was studied using etiolated leek seedling microsomes. In the presence of ATP, [2-¹⁴C]malonyl-CoA was incorporated into fatty acids of C₁₆C₂₆. The omission of ATP, even in the presence of acetyl-CoA, led to a complete loss of activity, which was restored by addition of exogeneous acyl-CoAs. Comparison of acyl-CoA (C₁₂C₂₄) elongation showed that stearoyl-CoA, in the presence of [2-¹⁴C]malonyl-CoA, was the more efficient precursor leading to the formation of fatty acids having a chain length of C₂₀C₂₆. [1-¹⁴C]C₁₆CoA and [1-¹⁴C]C₁₈CoA were elongated in the presence of malonyl-CoA, without degradation of the acyl chain. The time-course and the malonyl-CoA concentration curves showed that [1-¹⁴C]C₁₈CoA was a better primer than [1-¹⁴C]C₁₆CoA. Acyl-CoA elongation was also studied over the concentration range 4.5–45 μM [1-¹⁴C]C₁₈CoA. Comparison of the radioactivity incorporated into the fatty acids formed using [2-¹⁴C]malonyl-CoA in the presence of C₁₈CoA, on the one hand, and [1-¹⁴C]C₁₈CoA in the presence of malonyl-CoA, on the other, demonstrated clearly that the acyl chain of the acyl-CoA was elongated by malonyl-CoA.     

Alkaline decomposition of d-xylose-1-C, d-glucose-1-C, and d-glucose-6-C

The distribution of radioactivity in the three- and four-carbon saccharinic acids, lactic acid and 2,4-dihydroxybutyric acid, obtained from d-xylose-1-¹⁴C, d-glucose-1-¹⁴C, and d-glucose-6-¹⁴C, was measured. The relative importance of the various mechanisms for forming 2,4-dihydroxybutyric acid was determined. Recombination of two-carbon fragments was found to be an important mechanism at the high alkalinity and temperature employed.     

The biosynthesis of angelic acid in Cynoglossum officinale

Six-month-old Cynoglossum officinale plants were fed via the roots with tiglic acid-[1-¹⁴C]. After a week the plants were harvested, the alkaloid heliosupine was isolated and hydrolysed to heliotridine and angelic acid. The latter contained all the radioactivity of the original heliosupine showing that angelic acid may be formed from tiglic acid by a cis-trans isomerization.     

Metabolism of ent-kaurenol-[17-14C], ent-kaurenal-[17-14C] and ent-kaurenoic acid-[17-14C] by germinating Hordeum distichon grains

Subcellular fractions from germinated barley embryos, chloroplast preparations and whole germinating barley grains are able to carry out the conversions ent-kaurenol → ent-kaurenal → ent-kaurenoic acid → ent-hydroxykaurenoic acid, the initial steps of the biosynthetic pathway to gibberellins. Whole grains, and chloroplasts to a slight extent, incorporate radioactivity from ent-kaurenol-[17-¹⁴C] and ent-kaurenoic acid-[17-¹⁴C] into materials with similar but distinct properties from the gibberellins GA₁, GA₃, GA₄ and GA₇.     

The effect of 2-thiouracil on RNA synthesis in pollen tubesof Nicotiana alata

The level of RNA in pollen is approximately 20 mg g^-1 and remains constant during 6 h pollen germinationin vitro also in the presence of 2-thiouracil which stimulates pollen tube elongation. The synthesis of RNA in pollen tubes was investigated according to the incorporation of the label from uracil-2-¹⁴C, 2-thiouracil-2-¹⁴C, orotic acid-5-³H, fructose-U-¹⁴C and from³²PO₄ ^3- into RNA fractions separated by methylated albumine kieselguhr chromatography. The distribution of radioactivity on elution profiles was different according to the radioactivity source, however it was not changed by the presence of 2-thiouracil in cultivation medium. 2-Thiouracil incorporates into pollen tube RNA at about 50% the rate of uracil. It inhibited the incorporation of orotic acid, of fructose and of phosphate into all RNA fractions. It is suggested that the analogue inhibits the enzymes involved in RNA synthesis essentially as 2-thiouridine-5’-phosphate.     

Conversion of geranylgeranyl pyrophosphate to ent-kaurene in enzyme extracts of sonicated chloroplasts

Farnesyl pyrophosphate-[¹⁴C] and geranylgeranyl pyrophosphate-[¹⁴C] were biosynthesized from mevalonic acid-[2-¹⁴C] by cell-free enzyme extracts of pea (Pisum sativum) cotyledons containing MgCl₂, MnCl₂, ATP and AMO-1618. Maximum yields of farnesyl pyrophosphate were obtained after 30 min incubation while geranylgeranyl pyrophosphate was the primary product after 180 min. Biosynthesized geranylgeranyl pyrophosphate-[¹⁴C] served as an efficient substrate for ent-kaurene biosynthesis in reaction mixtures containing cotyledon enzymes when AMO-1618 was omitted. Enzyme extracts from green pea shoot tips and chloroplasts also converted geranylgeranyl pyrophosphate to ent-kaurene in very low yields. Ent-kaurene production from mevalonic acid-[2-¹⁴C] in extracts of pea shoot tips was also enhanced by addition of chloroplast enzymes. This evidence indicates that kaurene synthetase is present in pea chloroplasts and adds to the possibility that some gibberellin biosynthesis may be compartmentalized in those organelles.     

The Interrelationship of 2,4-Dichlorophenoxyacetic Acid with Molecular Components of the Cell during Callus Induction

This study investigates the fate of ¹⁴C labelled 2,4-D used as a trigger compound during callus induction (dedifferentiation). It also traces the special relationship of 2,4-D with some high molecular weight components in vivo, in which 2,4-D-2-¹⁴C was found combined with high molecular weight component-fractions. Nucleic acids showed very low radioactivities, based on MAK column chromatography and centrifugal profiles from sucrose density gradient treatments, but there was no specificity in this radioactive incorporation. Centrifugal profiles and Pronase treatment indicate that protein combines with 2, 4-D-2-¹⁴C. These results support the claim for a model of auxin-induced derepression, in which auxin interacts with its binding protein.     

Action of 2,4-DB and dalapon on the symbiotic properties ofLotus corniculatus (birdsfoot trefoil)

Summary Field trials carried out in 1965 and 1966 showed that 2,4-DB, alone or in combination with dalapon, reduced nodulation and tended to decrease the efficiency of nitrogen fixation in birdsfoot trefoil. Dalapon appeared to enhance the inhibitory action of 2,4-DB on nodulation. No obvious cytological differences could be detected in the nodules or in the isolated bacteroids of field-treated and untreated plants. Under growth chamber conditions, 2,4-DB drastically reduced trefoil growth and nodulation particularly in treatments where the herbicide came directly in contact with the plants. It appears that the reduction in nodulation and nitrogen fixation is a result of plant damage and abnormal root growth caused by 2,4-DB application.Autoradiographs indicated that the translocation of the herbicide was rapid, with detectable concentrations observed in young leaves, leafveins, roots, and nodules 12 hours after leaf-feeding of 2,4-DB-1-C¹⁴. The radio-activity appeared to accumulate with time (up to 5 days) in the growing root tips and nodules. Fractionation of excised nodules from trefoil plants demonstrated the presence of radioactivity in the cell debris, bacteroids, 29,000g pellet, plant ribosomes, and the soluble portion. The greatest accumulation of radioactivity occurred in the soluble fraction.The degradation of 2,4-DB and 2,4-D in trefoil was demonstrated by the evolution of C¹⁴O₂ from non-nodulated and aseptically growing plants leaf-fed with 2,4-DB-1-C¹⁴ or 2,4-D-1-C¹⁴.4-(2,4-dichlorophenoxy) butyric acid.2,2 dichloropropionic acid.     

Time-course tracer studies on the metabolism of cinnamic acid in Cestrum poeppigii

Time-course tracer studies were performed on the metabolism of trans-cinnamic acid-[3-¹⁴C] and trans-p-coumaric acid-[2-¹⁴C] in the     

The metabolism of [3−3H]oleanolic acid-3-O-mono-[14C]glucoside in isolated cells from Calendula officinalis leaves

The radioactive precursor, [3?³H]oleanolic acid-3-O-mono-[¹⁴C]glucoside was administrated to isolated cells obtained from the leaves of Calendula officinalis. The radioactivity of the precursor was incorporated into fractions containing free oleanolic acid, individual glucosides, glucuronide F and other glucuronides. The ratio of ³H: ¹⁴C radioactivity in these fractions indicated that glucosides were formed in a process involving direct glycosylation of the precursor, whereas the glucuronides were formed from oleanolic acid released by hydrolysis of the precursor. Dynamics curves showed that glucoside II formed by direct glycosylation of the precursor was intensively transformed to other derivatives.     

The Site of Synthesis of Sucrose in Green Plant Cells

Glycollic acid-1-¹⁴C, glyoxylic acid-2-¹⁴C, glycine-1-¹⁴C, andglycine-2-¹⁴C were fed to illuminated excised leaves of Pisumsativum and the distribution of ¹⁴C determined in the glycine,serine, sucrose, and insoluble polyglucan formed. Carboxyl-labelledglycollic acid and glycine gave rise to randomly labelled polyglucanand sucrose although the serine formed was predominantly carboxyllabelled. By contrast glyoxylic acid and glycine labelled inthe -carbon did not give rise to randomly labelled serine, sucrose,or polyglucan.     

Formation of 12-oxo-trans-10-dodecenoic acid in chloroplasts from Thea sinensis leaves

Linolenic acid-[1-¹⁴C] was converted to 12-oxo-trans-10-dodecenoic acid, via 12-oxo-cis-9-dodecenoic acid by incubation with chloroplasts of Thea sinensis leaves. Thus, it was confirmed that linolenic acid is split into a C₁₂-oxo-acid, 12-oxo-trans-10-dodecenoic acid, and a C₆-aldehyde, trans-2-hexenal, leaf aldehyde, by an enzyme system in chloroplasts of tea leaves.     

The interaction of carbon-14-labelled alkyl carbamates, labelled in the alkyl and carbonyl positions, with DNA in vivo

The binding of ethyl carbamate labelled with carbon-14 in the alkyl or carbonyl group, and of methyl, n-butyl and n-propyl carbamates labelled in the alkyl group, to the DNA of mouse liver, lung and kidney has been studied in male Crackenbush mice. Only ethyl carbamate bound to liver and kidney DNA to any significant extent.The binding of ethyl carbamate labelled with carbon-14 in the C₁, C₂ or the carbonyl position was examined and compared. The levels of binding of [1-¹⁴C]- and [2-¹⁴C]ethyl carbamate to liver DNA were not significantly different (328 ± 34 and 267 ± 24 dpm/mg DNA, respectively), but there was very little binding of the [carbonyl-¹⁴C]ethyl carbamate (26 ± 3 dpm/mg DNA). Furthermore, only 18% of the radioactivity was removed from the DNA labelled with the alkyl-labelled carbamates, whereas 65% of the radioactivity was removed from the DNA labelled with carbonyl-labelled ethyl carbamate on continuous ether extraction. It was concluded that the bound molecule does not contain the carbonyl carbon and is probably an ethyl group.