2-Trans-Abscisic Acid Biosynthesis and Metabolism of ABA-Aldehyde and Xanthoxin in Wild Type and the aba Mutant of Arabidopsis thaliana

Abscisic acid (ABA) and 2-trans-ABA (t-ABA) biosynthesis werestudied in wild type Landsberg erecta and the three allelicaba mutants of Arabidopsis thaliana (L.) Heynh., which are impairedin epoxy-carotenoid biosynthesis. Labelling experiments with¹⁸O₂and mass spectrometric analysis of [¹⁸O]ABA and its catabolitesABA-glucose ester (ABA-GE) and phaseic acid (PA), and t- ABAand t-ABA-GE, showed that t-ABA biosynthesis was less affectedthan ABA biosynthesis by mutations at the ABA locus. The aba-4allele caused the most severe impairment of ABA biosynthesiscompared with the other two mutant alleles aba-1 and aba-3,yet aba-4 plants synthesized as much t-ABA as wild type Landsbergerecta plants. Feeding experiments with RS- [²H₆]ABA-aldehydeisomers and unlabelled xanthoxin isomers suggest that t-xanthoxinand t-ABA-aldehyde are precursors to ABA and t-ABA in Arabidopsis Key words: ABA-alcohol, ABA-aldehyde, ABA-glucose ester, ¹⁸O₂ labelling, phaseic acid     

Precursors of Asparagine in Lupins

Fumaric acid-1,4-¹⁴C, L-aspartic acid-4-¹⁴C, and glycine-2-¹⁴Cwere supplied for 1 to 3 h to etiolated lupin shoots that wererapidly synthesizing asparagine. The distribution of ¹⁴C inaspartate and asparagine isolated from this material was determined.With aspartic acid-4-¹⁴C adminstration radioactivity was mostlyin carbons 1 and 4 with carbon 4 having the greater amount;with fumaric acid 1,4-¹⁴C administration, radioactivity wasagain mostly in carbons 1 and 4. Aspartate labelled by fumaratecontained relatively more radioactivity in carbons 2 and 3.Little radioactivity from glycine-2-¹⁴C entered either aspartateor asparagine and this was mainly in carbon 2 and 3. It is concludedthat asparagine synthesis form aspartate is a major pathwayof asparagine biosynthesis in lupins.     

Biosynthesis of Caffeine in Flower Buds of Camellia sinensis

The biosynthesis of purine alkaloids in flower buds of tea plantswas investigated. More than 25% of total radioactivity of [8-¹⁴C]adeninetaken up by stamens isolated from tea flower buds was foundto have been incorporated into purine alkaloids, namely, theobromineand caffeine, 24 h after administration of the labelled compound.Pulse-chase experiments indicated that [8-¹⁴C]adenine takenup by the stamens was converted to adenine nucleotides and subsequentlyincorporated into theobromine and caffeine. Since 5 µMcoformycin, an inhibitor of AMP deaminase, inhibited the incorporationof radioactivity into the purine alkaloids, synthesis of caffeinefrom adenine nucleotides seems to be initiated by the reactionof AMP deaminase. Although most of the radioactivity from [8-¹⁴C]inosinewas recovered as CO₂ and ureides, considerable amounts of radioactivitywere recovered as purine alkaloids. The incorporation of radioactivityfrom [8-¹⁴C]inosine into the purine alkaloids was not affectedby coformycin. The five enzymes involved in synthesis of 5-phosphoribosyl-1-pyrophosphatefrom glucose were present in the stamens and petals of tea flowerbuds. From present and previous results, the pathway for thebiosynthesis of caffeine from adenine nucleotides in flowerbuds of tea is discussed.Copyright 1993, 1999 Academic Press Camellia sinensis, tea, stamen, flower, biosynthesis, purine alkaloids, caffeine, theobromine, adenine nucleotides, nucleotide biosynthesis     

Compensatory Aspects of the Biosynthesis of Spermidine in Tobacco Cells in Suspension Culture

The relationship between the biosynthesis of polyamines andethylene was examined in suspension cultures of Nicotiana tabacumL. cells. Aminooxyacetic acid (AOA), an inhibitor of 1-aminocyclopropane-1-carboxylicacid synthase, inhibited the production of ethylene and raisedlevels of spermidine by increasing the availability of S-adenosylmethionine(SAM) for the synthesis of polyamines. In contrast, methylglyoxalbis (guanylhydrazone) (MGBG), an inhibitor of S-adenosylmethioninedecarboxylase (SAMDC), an enzyme involved in the biosynthesisof polyamines, caused a slight increase in the rate of biosynthesisof ethylene. However MGBG did not decrease the rate of biosynthesisof polyamines in 10-day-old senescing cells. Although MGBG inhibitedthe conversion of L-[U-^l4C]methionine into labeled spermidinevia SAM both in 4-day-and in 10-day-cultured cells, it stimulatedthe conversion of L-[U-^l4C]aspartic acid into labeled spermidinein 10-day-cultured cells. In actively dividing 4-day-culturedcells, L-[U-¹⁴C]homo-serine was also converted into polyamines.In senescing cells, which produce large amounts of ethylene,the biosynthesis of spermidine from aspartic acid coincidedwith that from methionine. In actively growing cells, whichproduce large amounts of polyamines, the biosynthesis of spermidinefrom homoserine coincided with that from methionine. These resultsindicate that homoserine and aspartic acid can be both usedas precursors in the biosynthesis of polyamines and help tomaintain appropriate titers of polyamines, when SAMDC is inhibitedand the level of decarboxylated SAM becomes limiting. (Received May 14, 1990; Accepted March 11, 1991)     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens II. IAA Biosynthetic Activity of Cultured Carrot Tissues Transformed with Wild-Type and Mutant Ti Plasmids

IAA biosynthetic activity was examined in cultured carrot tissuestransformed with Agrobacterium tumefaciens harboring wild-type,aux^– or cyt^– Ti plasmids. In vitro IAAM hydrolaseactivities in tissues transformed with wild-type, and cyt^–Ti plasmids were 3.09 and 19.82 nmol/g proteins/30 min, respectively,but not detectable when aux^– Ti plasmids were used. Theactivity of IAA biosynthesis, determined by the incorporationof radioactivity into IAA in tissues fed with [¹⁴C]-tryptophan,was 34.13, 10.92 and 32.47 pmol/g fr wt/30 min in tissues transformedwith wild type, aux^– and cyt^– Ti plasmids, respectively.The incorporation of radioactivity into the IAAM fraction wasdetected only in the tissues transformed with wild type andcyt^– Ti plasmids. These results showed that the T-DNAencoded pathway of IAA biosynthesis was active in tissues transformedwith wild-type and cyt^h Ti plasmids, and that the activity ofIAA biosynthesis in those tissues was higher than that in tissuestransformed with the aux^– Ti plasmid. (Received March 16, 1988; Accepted July 31, 1988)     

L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

We investigatedthe role of intracellular calcium concentration([Ca²⁺]_i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca²⁺]_i, and the presence of L-typevoltage-dependent Ca²⁺ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca²⁺-chelatingagent EDTA (100 mM), the L-type Ca²⁺ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca²⁺ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca²⁺channels in endothelial cells, the [Ca²⁺]_isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca²⁺]_i. Pretreatment ofendothelial cells with high K⁺ (60 mM) or nifedipine (4 µM) diminished increases in [Ca²⁺]_i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca²⁺]_i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca²⁺]_i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca²⁺ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca²⁺ channels.

     

Lipids in Cryptomonas CR-1. II. Biosynthesis of Betaine Lipids and Galactolipids

The biosynthesis of lipids in Cryptomonas strain CR-1 was studiedusing radioactive tracers. For studies of general aspects ofthe biosynthesis of lipids, the cells were labelled with [¹⁴C]NaHCO₃or with [l,3-¹⁴]glycerol. In both cases, monogalactosyl diacylglycerol(MGDG) was the most heavily labelled lipid. Phosphatidylcholineand the alanine lipid DGTA were not labelled to specific activitiescomparable to those of MGDG and DGDG. It is improbable thatthe so-called "eukaryotic pathway", which has been suggestedas the pathway for the synthesis of " eukaryotic" molecularspecies of MGDG from PC in higher plants, is operative in Cryptomonascells which contain typical "eukaryotic" MGDG. The homoserinelipid DGTS was labelled to a significant level only in its polargroup. The C-3 and C-4 atoms of methionine, as well as the methylcarbon of methionine, were incorporated into both DGTS and DGTA,whereas the C-l carbon of methionine was incorporated uniquelyinto DGTS. Results of pulse-chase experiments with [3,4-¹⁴C]methionineand [methyl.-^l4C]methionine suggest the conversion of DGTS toDGTA. (Received April 22, 1991; Accepted June 12, 1991)     

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.     

Nudeotide Pools and Purine Metabolism in Tissue Cultures of Wild-Type Datura innoxia and Adenine-Requiring Auxotroph

Cells of the auxotrophic mutant, Ad1, of Datura innoxia requiredadenine, adenosine, or inosine for their growth on solid agarmedium which contained Murashige-Skoog salts, 2,4-dichloro-phenoxyaceticacid, and sucrose. Thirteen purine and pyrimidine nucleotidesin extracts of wild-type and Ad1 cells were separated and quantifiedby HPLC. Levels of ADP-glucose and UMP were significantly higherin Ad1 than in wild-type cells, but those of other nucleotideswas found when Ad1 cells were transferred to fresh medium withoutadenine. The rate of the biosynthesis de novo of purines, asestimated from the rate of incorporation of ¹⁴C from [2-¹⁴C]-glycine and [¹⁴C]formate into adenine nucleotides, was reducedin Ad1 cells to 21 and 13% of the wild-type rate, respectively.The activities involved in the salvage of adenine and adenosinein Ad1 cells were similar to those in wild-type cells. Ad1 cellshad the capability to convert adenine to guanine nucleotidesand guanine to adenine nucleotides. ¹ Part 27 of the series, "Metabolic Regulation in Plant CellCulture". (Received March 7, 1988; Accepted August 3, 1988)     

Enzymes involved in the last steps of the biosynthesis of mannitol in brown algae

Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17 [EC] ) and mannitol-1-phosphatase(EC number yet unassigned) were detected in the brown algae,Spatoglossum pacificum and Dictyota dichotoma. The enzymes wereextracted from algal fronds and their properties were investigatedusing partially purified preparations. Mannitol-1-phosphatase shows maximum activity at pH 7. The enzymehad a narrow substrate specificity. The Km value for mannitol-1-phosphateis 8.3x10^–4 M (30°C, pH 7.0). The enzyme is activatedby Mg⁺⁺ and Mn⁺⁺and is strongly inhibited by PCMB, Hg⁺⁺and NaF. Mannitol-1-phosphate dehydrogenase showed maximum activitiesat pH values 6.5 and 10.2 in reductive and oxidative reactions,respectively. The dehydrogenase also showed narrow substratespecificity; mannitol-1-phosphate and NAD or fructose-6-phosphateand NADH₂ are utilized, respectively, in oxidative and reductivereactions by the enzyme. Km values for these substrates andthe coenzymes are 2.5x10^–4 M and 7.1x10^–5 M forthe first pair and 2.8x10^–4 M and 1.3x10^–5 M forthe latter pair. This enzyme was strongly inhibited by PCMBand Hg⁺⁺, but was only slightly affected by adenosine phosphates. Possible roles of these enzymes in the biosynthesis of mannitolin brown algae are discussed. ¹ Contributions from the Shimoda Marine Biological Station ofTokyo Kyoiku University, No. 233. This work was supported inpart by a Grant-in-Aid for Co-operative Research from the Ministryof Education, Japan and in part by a grant to one of us (T.Ikawa) from the Matsunaga Science Foundation. ² Present address: Chemical and Physical Laboratory, HoechstJapan Research Laboratory, Minamidai, Kawagoe, Japan. (Received February 22, 1972; )     

Effect of Inorganic Phosphate on the Biosynthesis of Purine and Pyrimidine Nucleotides in Suspension-Cultured Cells of Catharanthus roseus

The levels of purine and pyrimidine nucleotides in suspensioncultures of Catharanthus roseus were determined 24 h after stationary-phasecells were transferred to fresh complete (‘+Pi’)or phosphate-deficient (‘–Pi’) Murashige-Skoogmedium. The levels of ATP, GTP, UTP and CTP were from approx.3 to 5-fold greater in the cells grown in ‘+Pi’medium than in the cells grown in ‘–Pi’ medium.The levels of almost all other nucleotides were slightly higherin the cells in ‘+Pi’ medium. The rates of de novoand salvage biosynthesis of purine and pyrimidine nucleotideswere estimated from the rates of incorporation of radioactivityfrom [¹⁴C]formate, [2–¹⁴C]glycine, NaH¹⁴CO₃, [6–¹⁴C]orotate,[8–¹⁴C]adenine, [8–¹⁴C]adenosine, [2–¹⁴C]uraciland [2–¹⁴C]uridine. The results indicated that the activityof both the de novo and the salvage pathway was higher in thecells in ‘+Pi’ medium than in the cells in ‘–Pi’medium. The rate of degradation estimated from the rate of releaseof ¹⁴CO₂ from labelled purines and pyrimidines indicated thatdegradation of uridine was significantly reduced in the cellsin ‘+Pi’ medium, but no significant difference wasfound in the degradation of adenine, adenosine and uracil. Thepossible role of Pi in the control of the biosynthesis of nucleotidesand in the degradation of uridine is discussed. Catharanthus roseus, Madagascar periwinkle, suspension culture, inorganic phosphate, nucleotides, purines, pyrimidines, biosynthesis, degradation     

Biosynthesis of Cytokinin in Germinating Seeds of Zea mays

The embryos of germinating Zea mays seed were supplied with[¹⁴C]-adenine Following incubation, the tissue was extractedand extensively purified by non-exchange chromatography andthin layer chromatography. Radioactivity was found to be incorporatedinto zeatin nucleotide indicating that the embryo in the germinatingseed is capable of cytokinin biosynthesis. Key words: Zea mays cytokinin, zeatin nucleotide, biosynthesis, seed     

Regulation of Sterol Biosynthesis in Solanum Species

Regulation of sterol synthesis was studied in Solanum species.A significant negative correlation was found between sterolcontent and rate of sterol synthesis from (1-¹⁴C) acetate inplant organs of Solanum nigrum and cell cultures of S. dulcamara.Exogenous cholesterol significantly inhibited the rate of sterolsynthesis from (¹⁴C) acetate in cell cultures of S. dulcamarawithout affecting synthesis from (³H) mevalonate. Exogenouscholesterol stimulated the rate of total lipid synthesis fromboth (¹⁴C) acetate and (³H) mevalonate. Thus, cholesterol inhibitedconversion of acetate to mevalonate; this is taken as evidenceof a negative feedback control on sterol synthesis. Key words: Feedback control, Phytosterol biosynthesis, Plant cell culture, Solanum species     

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 Biosynthesis of Cytokinins in Germinating Lupin Seeds

Imbibed intact seeds, and excised embryos and cotyledons ofyellow lupin (Lupinus luteus L. cv. Weiko III) have been incubatedwith [¹⁴C]-adenine to investigate cytokinin biosynthesis duringthe early stages of germination. Following incubation the tissueswere extracted and purified by solvent partition and chromatographyon cellulose phosphate, diethylaminoethyl cellulose and SephadexLH-20 columns. Using a variety of thin layer chromatographic,high performance liquid chromato-graphic and chemical procedures,incorporation of ¹⁴C into dihydrozeatin riboside and its nucleotidewas demonstrated in extracts of intact embryos, intact cotyledonsand excised embryos. However, radioactivity was not found associatedwith cytokinins in fractions derived from the isolated cotyledons.This is the first direct demonstration of cytokinin biosynthesisin germinating seeds and the results indicate that the capacityfor cytokinin biosynthesis is probably confined to the embryonicaxes. If this is so, the levels of [¹⁴CJ-dihydrozeatin ribosideassociated with intact embryo and intact cotyledon fractionsindicate that the synthesized cytokinin is transported to andaccumulates in the cotyledons. Key words: Lupinus luteus, cytokinin biosynthesis, seed germination     

Unsaponifiable Lipid Biosynthesis in the Seedling of Euphorbia lathyris L.: A Bypass via Alcoholic Fermentation

[1-¹⁴C]-ethanol supplied to the cotyledons of 9-d-old Euphorbialathyris seedlings was rapidly incorporated into unsaponifiablelipids, particularly into sterols, latex triterpenols and intothe triterpene ketones of the epicuticular wax. The [¹⁴C]-triterpenoidproduction from ethanol was hardly affected by sucrose in theexternal medium when sucrose uptake rates were low, but whenthe uptake rate was higher the [¹⁴C]-triterpenoid productionfrom [¹⁴C]-ethanol was greatly reduced. This observation isconsistent with the proposition that at high sucrose uptakerates, some sucrose is converted into ethanol, so that the incorporationof [¹⁴C]-ethanol into triterpenoids is reduced by competitionwith endogenously formed ethanol. A calculation based on theputative daily ethanol production in the cotyledons and thedaily triterpenoid production of seedlings indicates that about10 % of the triterpenoid synthesis in vivo may be from ethanol. Ethanol, Euphorbia lathyris, fermentation, seedling, triterpenoid biosynthesis     

Biosynthesis and partitioning of individual glucosinolates between pod walls and seeds and evidence for the occurrence of PAPS:desulphoglucosinolate sulphotransferase in seeds of oilseed rape (Brassica napus L.)

³⁵SO₄^2–; and ³⁵S-labelled glucosinolate precursors wereadministered to intact whole-pods and seeds to investigate thecapacity of oilseed rape (Brassica napus L.) pod tissues tocarry out reactions of the glucosinolate biosynthetic pathway.³⁵S-desulphobut-3-enyl and ³⁵S-desulphoindol-3-ylmethyl glucosinolateswere converted to their sulphonated ‘intact glucosinolate’homologues by isolated immature seeds. A neutral sulphur-containingfraction was isolated from pod walls and shown to be associatedwith glucosinolate biosynthesis. Further purification of thisfraction showed the presence of desulphoglucosinolates, thepenultimate intermediates in the glucosinolate biosyntheticpathway. Chemical characterization and quantification of theseintermediates showed that their types and levels correspondedto the glucosinolate biosynthetic activity of pod-wall tissues.‘Partition quotients’ (Pq) were calculated for individualglucosinolates from ³⁵S-labelling data and used to describethe apportionment of newly synthesized glucosinolates betweenpod walls and seeds. Results from continuous feeding studieswith pods and ³⁵SO₄^2–; indicated that individual rapeseedglucosinolates have characteristic Pq values. Key words: biosynthesis, desulphoglucosinolates, glucosinolates, partitioning, rapeseed     

Gibberellins, Amylase, and the Onset of Heterosis in Maize Seedlings

Rood, S. B. and Larsen, K. M. 1988. Gibberellins, amylase, andthe onset of heterosis in maize seedlings.—J. exp. Bot.39: 223–233. The possible involvement of gibberellins and amylase in heterosisof maize seedlings was investigated in two parental inbreds,CM7 and CM49, and their single cross F₁ hybrid, CM7xCM49. Germinationof all three genotypes was complete within 36 h after the onsetof imbibition. By 48 h, heterosis (hybrid vigour) for increasedshoot and root length was consistently observed. The endogenousconcentration of gibberellin A₁ (GA₁) was measured in 48 h seedlingsby gas chromatography-mass spectrometry with selected ion monitoring(GC-SIM) using [²H₂]-GA₁ as an internal standard. The GA₁ concentrationwas highest in the hybrid (59 ng g^–1 dry wt.), intermediatein CM49 (9.0 ng g^–1), and lowest in CM7 (<5.0 ng g^–1).Amylase activities in all three genotypes were very low at 24h, but increased during the next 24 h, after which time amylaseactivity in the hybrid was significantly higher than that ofeither parental inbred. Inhibitors of gibberellin (GA) biosynthesis,AMO-1618 or CCC, inhibited germination, shoot and root growth,and amylase activity in all three genotypes. Conversely, exogenousgibberellic acid (GA₃) increased amylase activity, particularlyin the inbred CM7. Amylase isozymes were separated through polyacrylamidegel electrophoresis and generally similar profiles of starchdegrading enzymes were observed in the three genotypes. SinceGA is known to control a-amylase biosynthesis in some cereals,these results are consistent with the hypothesis that GAs areinvolved in the regulation of heterosis in maize. A higher endogenousGA₁ concentration in the hybrid could result in increased amylaseactivity in the hybrid seedlings and consequently, more rapidstarch hydrolysis which fuels heterosis for early growth. Key words: Amylase, germination, gibberellic acid, Gibberellin A₁, heterosis, hybrid vigour, Zea mays