Biosynthesis, translocation, and accumulation of betaine in sugar beet and its progenitors in relation to salinity

Like other halophytic chenopods, sugar beet (Beta vulgaris L.) can accumulate high betaine levels in shoots and roots. N,N,N-trimethylglycine impedes sucrose crystallization and so lowers beet quality. The objective of this research was to examine the genetic variability and physiological significance of betaine accumulation in sugar beet and its relatives. Three cultivated genotypes of B. vulgaris and two genotypes of the wild progenitor B. maritima L. were grown with and without gradual salinization (final NaCl concentration = 150 millimolar). At 6 weeks old, all five genotypes had moderately high betaine levels in shoots and roots when unsalinized (averages for all genotypes: shoots = 108 micromoles per gram dry weight; roots = 99 micromoles per gram dry weight). Salinization raised betaine levels of shoots and roots 2- to 3-fold, but did not greatly depress shoot or root growth. The genotype WB-167—an annual B. maritima type—always had approximately 40% lower betaine levels in roots than the other four genotypes, although the betaine levels in the shoots were not atypically low.

The site and pathway of betaine synthesis were investigated in young, salinized sugar beet plants by: (a) supplying 1 micromole [¹⁴C]ethanolamine to young leaf blades or to the taproot sink of intact plants; (b) supplying tracer [¹⁴C]formate to discs of leaf, hypocotyl, and taproot tissues in darkness. Conversion of both ¹⁴C precursors to betaine was active only in leaf tissue. Very little ¹⁴C appeared in the phospholipid phosphatidylcholine before betaine was heavily labeled; this was in marked contrast to the labeling patterns in salinized barley. Phosphorylcholine was a prominent early ¹⁴C metabolite of both [¹⁴C]ethanolamine and [¹⁴C]formate in all tissues of sugar beet. Betaine translocation was examined in young plants of sugar beet and WB-167 by applying tracer [methyl-¹⁴C]betaine to a young expanded leaf and determining the distribution of ¹⁴C after 3 days. In all cases, extensive ¹⁴C translocation to young leaves and taproot sink occurred; neither in the fed leaf nor in sink organs were any ¹⁴C metabolites of betaine detected.

     

The incorporation of monomethylethanolamine and dimethylethanolamine in fetal brain aggregating cell culture

Fetal rat brain aggregating cell cultures were exposed to varying concentrations of [³H]monomethylethanolamine (MME) and [³H] dimethylethanolamine (DME). The rate of labeling of water-soluble compounds was more rapid and the amount of radioactivity present was greater than in the lipids. After a 72 hour incubation in the presence of millimolar concentrations of these nitrogenous bases, the major water-soluble products were the phosphorylated form of the bases. Little label was associated with the free bases or their cytidyl derivate. In the phospholipids, 97% of the radioactivity was recovered in phosphatidylmonomethylethanolamine (PMME) and 3% in phosphatidyldimethylethanolamine (PDME) or 95% in PDME and 5% in phosphatidylcholine (PC) after growth in presence of [³H]MME and [³H]DME respectively. The rate of formation of the radioactive products increased as function of the concentration of the nitrogenous base added up to 4 mM, the highest concentration employed. There was no significant difference in the pattern of labeling with cells grown in media devoid of methionine or choline. The turnover of the water-soluble metabolites was more rapid than in the phospholipids where an apparent half-life of 24 hours was calculated.Abbreviations PMT phospholipid-N-methyltransferase - AdoMet S-adenosyl-L-methionine - EA ethanolamine - MME N-monomethylethanolamine - DME N,N-dimethylethanolamine - CH choline - PE phosphatidylethanolamine - PMME phosphatidylmonomethylethanolamine - PDME phosphatidyldimethylethanolamine - PC phosphatidylcholine - PS phosphatidylserine - CAPS cyclohexylaminopropane sulfonic acid     

Digestion of phosphatidylcholines,absorption, and esterification of lipolytic products by Aeshna cyanea larvae as studied in vivo and in vitro

Digestion and absorption of phosphatidylcholine by Aeshna cyanea larvae were studied in vivo and in vitro with the isolated digestive juice and isolated midgut. The experiments were performed with stable ether analogues (1-alkyl-2-acyl-,1,2-dialkyl phosphatidylcholine, and 1-monoalkyl-lysophosphati-dylcholine), with radioactive 1,2-diacylphosphatidylcholine alternatively labelled in the acyl- and choline moieties, and with several phosphatidylcholine derivatives (1-[1-¹⁴C]acyl- and 1-[³H] alkyl-lysophosphatidylcholine, [1-¹⁴C]oleic acid, [2-¹⁴C]glycerol, phosphoryl[methyl-¹⁴C]choline, and [methyl-¹⁴C]choline). Chromatographic analyses of the digestion products revealed that phosphatidylcholine was degraded via two interconnected hydrolytic pathways involving phospholipase C, phospholipase A₂, lipase, and alkaline phosphatase. Complete hydrolysis by these pathways yielded the same four end products: free fatty acid, glycerol, choline, and P_i, which were absorbed by the midgut enterocytes. Of the intermediate hydrolysates, lysophosphatidylcholine, monoacylglycerol, and possibly phosphorylcholine were also absorbed. Radiolabelled oleic acid, glycerol, lysophosphatidylcholine and monoacylglycerol (as judged from monoalkylglycerol absorption) were incorporated into phospholipids and acylglycerols of the midgut enterocytes and were released into the haemolymph primarily in the form of diacylglycerols. In the case of glycerol ingestion, a small fraction of haemolymph radioactivity was associated with free glycerol and glycerolphosphate. After absorption by the enterocytes, radiolabelled choline was partly oxidized to betaine, partly phosphorylated, and partly incorporated into lyso- and phosphatidylcholine. It was recovered from the haemolymph predominantly as free choline, phosphorylcholine, and betaine. Arch. Insect Biochem. Physiol. 36:273–293, 1997. © 1997 Wiley-Liss, Inc.     

Betaine Accumulation and [C]Formate Metabolism in Water-stressed Barley Leaves

Barley (Hordeum vulgare L.) plants at the three-leaf stage were water-stressed by flooding the rooting medium with polyethylene glycol 6000 with an osmotic potential of −19 bars, or by withholding water. While leaf water potential fell and leaf kill progressed, the betaine (trimethylglycine) content of the second leaf blade rose from about 0.4 micromole to about 1.5 micromoles in 4 days. The time course of betaine accumulation resembled that of proline accumulation. Choline levels in unstressed second leaf blades were low (<0.1 micromole per blade) and remained low during water stress. Upon relief of stress, betaine-like proline—remained at a high concentration in drought-killed leaf zones, but betaine did not disappear as rapidly as proline from viable leaf tissue during recovery.

When [methyl-¹⁴C]choline was applied to second leaf blades of intact plants in the growth chamber, water-stressed plants metabolized 5 to 10 times more ¹⁴C label to betaine than control plants during 22 hours. When infiltrated with tracer quantities of [¹⁴C]formate and incubated for various times in darkness or light, segments cut from water-stressed leaf blades incorporated about 2- to 10-fold more ¹⁴C into betaine than did segments from unstressed leaves. In segments from stressed leaves incubated with [¹⁴C]formate for about 18 hours in darkness, betaine was always the principal ¹⁴C-labeled soluble metabolite. This ¹⁴C label was located exclusively in the N-methyl groups of betaine, demonstrating that reducing equivalents were available in stressed leaves for the reductive steps of methyl group biosynthesis from formate. Incorporation of ¹⁴C from formate into choline was also increased in stressed leaf tissue, but choline was not a major product formed from [¹⁴C]formate.

These results are consistent with a net de novo synthesis of betaine from 1- and 2-carbon precursors during water stress, and indicate that the betaine so accumulated may be a metabolically inert end product.

     

Radiotracer evidence implicating phosphoryl and phosphatidyl bases as intermediates in betaine synthesis by water-stressed barley leaves

In barley, glycine betaine is a metabolic end product accumulated by wilted leaves; betaine accumulation involves acceleration of de novo synthesis from serine, via ethanolamine, N-methylethanolamines, choline, and betaine aldehyde (Hanson, Scott 1980 Plant Physiol 66: 342-348). Because in animals and microorganisms the N-methylation of ethanolamine involves phosphatide intermediates, and because in barley, wilting markedly increases the rate of methylation of ethanolamine to choline, the labeling of phosphatides was followed after supplying [¹⁴C]ethanolamine to attached leaf blades of turgid and wilted barley plants. The kinetics of labeling of phosphatidylcholine and betaine showed that phosphatidylcholine became labeled 2.5-fold faster in wilted than in turgid leaves, and that after short incubations, phosphatidylcholine was always more heavily labeled than betaine. In pulse-chase experiments with wilted leaves, label from [¹⁴C]ethanolamine continued to accumulate in betaine as it was being lost from phosphatidylcholine. When [¹⁴C]monomethylethanolamine was supplied to wilted leaves, phosphatidylcholine was initially more heavily labeled than betaine. These results are qualitatively consistent with a precursor-to-product relationship between phosphatidylcholine and betaine.     

A conformational study of some adenosines by use of nuclear Overhauser effect

Conformations of 8-bromo-2′-[unk]-triisopropylbenzenesulfonyladenosine ([unk]) and its 3′-[unk]-isomer ([unk]) in solution have been determined by the use of intramolecular nuclear Overhauser effects in ¹H NMR spectroscopy. Compound [unk] has been proved to have a conformation in which the adenosine and benzene rings are intramolecularly stacked and compound [unk] an elongated non-stacked conformation in dimethylsulphoxide. The 5′-[unk]-acetyl derivative of [unk] has also been found to adopt the intramolecularly stacked conformation in dimethylsulphoxide, but a non-stacked one in chloroform. Coupling constants observed are discussed in connection with the conformation of the ribose moiety. The ¹³C NMR spectra have also been examined, but no effect which could be ascribed to the stacking phenomena was observed in the carbon chemical shifts.     

Choline Synthesis in Spinach in Relation to Salt Stress

Choline metabolism was examined in spinach (Spinacia oleracea L.) plants growing under nonsaline and saline conditions. In spinach, choline is required for phosphatidylcholine synthesis and as a precursor for the compatible osmolyte glycine betaine (betaine). When control (nonsalinized) leaf discs were incubated for up to 2 h with [1,2-14C]ethanolamine, label appeared in the N-methylated derivatives of phosphoethanolamine including phosphomono-, phosphodi-, and phosphotri- (i.e. phosphocholine) methyl-ethanolamine, as well as in choline and betaine, whereas no radioactivity could be detected in the mono- and dimethylated derivatives of the free base ethanolamine. Leaf discs from salinized plants showed the same pattern of labeling, although the proportion of label that accumulated in betaine was almost 3-fold higher in the salinized leaf discs. Enzymes involved in choline metabolism were assayed in crude leaf extracts of plants. The activites of ethanolamine kinase and of the three S-adenosylmethionine:phospho-base N-methyltransferase enzymes responsible for N-methylating phosphoethanolamine to phosphocholine were all higher in extracts of plants salinized step-wise to 100, 200, or 300 mM NaCI compared with controls. In contrast, choline kinase, phosphocholine phosphatase, and cytidine 5[prime]-triphosphate: phosphocholine cytidylyltransferase activities showed little variation with salt stress. Thus, the increased diversion of choline to betaine in salt-stressed spinach appears to be mediated by the increased activity of several key enzymes involved in choline biosynthesis.     

Phosphatidylglycerol synthesis in pea chloroplasts: pathway and localization

Isolated intact pea chloroplasts synthesized phosphatidylglycerol from either [¹⁴C]acetate or [¹⁴C]glycerol 3-phosphate. Both time-course and pulse-chase labeling studies demonstrated a precursor-product relationship between newly synthesized phosphatidic acid and newly synthesized phosphatidylglycerol.

The synthesis both of CDP-diacylglycerol from exogenous phosphatidic acid and CTP, and of phosphatidylglycerol from exogenous CDP-diacylglycerol and glycerol 3-phosphate, could be assayed in fractions obtained from disrupted chloroplasts. Moreover, the enzymes catalyzing these reactions were localized in the inner envelope membrane. Exogenous phosphatidic acid was incorporated into phosphatidylglycerol, but only following its incorporation into CDP-diacylglycerol. Finally, radio-active phosphatidic acid synthesized in the envelope membranes from [¹⁴C]palmitoyl-ACP and 1-oleoyl-glycerol 3-phosphate was sequentially incorporated into labeled CDP-diacylglycerol and phosphatidylglycerol upon the addition of appropriate substrates and cofactors. Thus, we have demonstrated that (a) the synthesis of phosphatidylglycerol in chloroplasts occurs by the pathway: phosphatidic acid → CDP-diacylglycerol →→ phosphatidylglycerol, and (b) phosphatidylglycerol synthesis is located in the inner envelope membrane.

     

Phosphatidylcholine Synthesis in Castor Bean Endosperm : Occurrence of an S-Adenosyl-l-Methionine:Ethanolamine N-Methyltransferase

The methylation steps in the biosynthesis of phosphatidylcholine by castor bean (Ricinus communis L.) endosperm have been studied by pulse-chase labeling. Endosperm halves were incubated with [methyl-¹⁴C]S-adenosyl-l-methionine, [2-¹⁴C]ethanolamine, [¹⁴C]ethanolamine phosphate, or [¹⁴C]serine phosphate. The kinetics of appearance were followed in the free, phospho-, and phosphatidyl-bases. The initial methylation utilized ethanolamine as a substrate to form methylethanolamine, which was then converted to dimethylethanolamine, choline, and phosphomethylethanolamine. Subsequent methylations occurred at the phospho-base and, to a lesser extent, the phosphatidyl-base levels, after which the radioactivity either remained constant or decreased in these compounds and accumulated in phosphatidylcholine. Although the precursors tested did support the synthesis of choline, the kinetics of the labeling make them unlikely to be the major sources of free choline to be utilized for the nucleotide pathway. A model with two pools of choline is proposed, and the implications of these results for the pathways leading to phosphatidylcholine biosynthesis are discussed.     

Intermediates in the recycling of 5-methylthioribose to methionine in fruits

The recycling of 5-methylthioribose (MTR) to methionine in avocado (Persea americana Mill, cv Hass) and tomato (Lycopersicum esculentum Mill, cv unknown) was examined. [¹⁴CH₃]MTR was not metabolized in cell free extract from avocado fruit. Either [¹⁴CH₃]MTR plus ATP or [¹⁴CH₃]5-methylthioribose-1-phosphate (MTR-1-P) alone, however, were metabolized to two new products by these extracts. MTR kinase activity has previously been detected in these fruit extracts. These data indicate that MTR must be converted to MTR-1-P by MTR kinase before further metabolism can occur. The products of MTR-1-P metabolism were tentatively identified as α-keto-γ-methylthiobutyric acid (α-KMB) and α-hydroxy-γ-methylthiobutyric acid (α-HMB) by chromatography in several solvent systems. [³⁵S]α-KMB was found to be further metabolized to methionine and α-HMB by these extracts, whereas α-HMB was not. However, α-HMB inhibited the conversion of α-KMB to methionine. Both [U-¹⁴C]α-KMB and [U-¹⁴C]methionine, but not [U-¹⁴C]α-HMB, were converted to ethylene in tomato pericarp tissue. In addition, aminoethoxyvinylglycine inhibited the conversion of α-KMB to ethylene. These data suggest that the recycling pathway leading to ethylene is MTR → MTR-1-P → α-KMB → methionine → S-adenosylmethionine → 1-aminocyclopropane-1-carboxylic acid → ethylene.     

The oxidation of choline by liver slices and mitochondria during liver development in the rat

Betaine is the major oxidation product of [Me-¹⁴C] choline produced by rat liver slices. Liver slices from adult rats rapidly oxidize [Me-¹⁴C] choline to betaine and the bulk of the betaine produced is recovered in the incubation medium. Considerably more choline is oxidized to betaine than is phosphorylated to phosphorylcholine. The rate of phosphorylation of choline appears to be independent of the rate of choline oxidation. Liver slices from fetal and young rats oxidize choline to betaine at a lower rate than adult liver slices.The ability of mitochondria to oxidize [Me-¹⁴C] choline to betaine aldehyde and betaine is considerably lower in fetal liver than in adult liver. The major product with both fetal and adult mitochondria is betaine aldehyde. Choline oxidation by mitochondria begins to increase 1 day prior to birth and increases progressively to adult levels by 18 days. The developmental pattern for choline oxidation is similar to the pattern for succinic dehydrogenase activity.     

Gramine Accumulation in Leaves of Barley Grown under High-Temperature Stress

The indole alkaloid gramine is toxic to animals and may play a defensive role in plants. Under certain conditions, shoots of barley cultivars such as `Arimar' and CI 12020 accumulate gramine (N,N-dimethyl-3-aminomethylindole) and lesser amounts of its precursors 3-aminomethylindole (AMI) and N-methyl-3-aminomethylindole (MAMI); other cultivars such as `Proctor' do not. When grown at optimal temperatures (21°C/16°C, day/night), Arimar contained a high level of gramine in the first leaf (approximately 6 milligrams per gram dry weight), but progressively less accumulated in successive leaves so that the gramine level in the shoot as a whole fell sharply with age. In Arimar and CI 12020 plants transferred at the two- to three-leaf stage from 21°C/16°C to supra-optimal temperatures (≥30°C/25°C), there was massive gramine accumulation in leaves which developed at high temperature, so that gramine level in the whole shoot remained high (about 3-8 milligrams per gram dry weight).

Proctor lacked both constitutive gramine accumulation in the first leaf and heat-induced gramine accumulation in later leaves. The following evidence indicates that this results from a lesion in the pathway of synthesis (tryptophan →→ AMI → MAMI → gramine) between tryptophan and AMI. (a) Proctor and Arimar leaves readily absorbed [¹⁴C]gramine, but neither cultivar degraded it extensively. (b) Arimar leaf tissue incorporated [¹⁴C]formate label into the N-methyl groups of gramine and MAMI, and converted [methylene-¹⁴C]tryptophan to AMI, MAMI, and gramine; Proctor leaf tissue did not, even when a trapping pool of unlabeled gramine was supplied. (c) Proctor converted [¹⁴C]MAMI to gramine as actively as Arimar. (d) Proctor incorporated [¹⁴C]formate label into gramine and MAMI when supplied with AMI; the ratio [¹⁴C]gramine/[¹⁴C]MAMI fell with leaf age, suggesting that the two N-methylations involve different enzymes. Inasmuch as Proctor leaf tissue did not methylate added tryptamine or tyramine, the N-methyltransferase(s) of gramine synthesis may be substrate specific.

In sterile culture at optimal temperatures, 10 millimolar gramine did not affect autotrophic growth of Arimar or Proctor plantlets or heterotrophic growth of callus. At supra-optimal temperature, plantlet growth was reduced by gramine although callus growth was not. We speculate that gramine-accumulating cultivars may suffer autotoxic effects at high leaf temperatures.

     

Increased catabolism of phosphatidylcholine to betaine regulates the liver phosphatidylcholine content in rats fed orotic acid

Feeding a semi-synthetic diet containing 1% orotic acid to rats for one day stimulates the CDPcholine pathway of liver phosphatidylcholine synthesis 4.5-fold without significantly increasing the liver phosphatidylcholine level. The liver betaine level increases 1.6-fold. The present experiments were performed to investigate the source of the increased liver betaine. Orotic acid feeding did not alter the rate of oxidation of 1,2[14C] choline to betaine. After liver phosphatidylcholine was labelled in vivo with 2[14-C]-ethanolamine, over 90% of the choline-derived radioactivity was recovered in liver betaine and this was consistently increased in rats fed orotic acid. It is concluded that the increased synthesis of liver phosphatidylcholine caused by dietary orotic acid is accompanied by an increased rate of liver phosphatidylcholine catabolism, with betaine as the major end-product of the choline moiety.     

Accumulation of glycinebetaine and its synthesis from radioactive precursors under salt-stress in the cyanobacterium Aphanothece halophytica

Growth in salt-stressed (2.0 M NaCl) Aphanothece halophytica was initially delayed during the first two days of cultivation and eventually attained the same growth rate as the control (0.5 M NaCl) cells. Glycinebetaine accumulation increased slightly in control cells but a dramatic increase of glycinebetaine occurred in salt-stressed cells during a growth period of six days. There was no apparent increase in the synthesis of [¹⁴C] glycinebetaine in the control cells, in contrast to the marked increase in its synthesis in the salt-stressed cells. Increasing NaCl concentration in the growth medium induced both the accumulation and the synthesis of glycinebetaine. Time course experiments provided evidence that [¹⁴C] choline was first oxidized to [¹⁴C] betaine aldehyde which was further oxidized to [¹⁴C] glycinebetaine in A. halophytica. The supporting data for such a pathway were obtained from the presence of choline and betaine aldehyde dehydrogenase activities found in the membrane and cytoplasmic fractions, respectively. The activities of these two enzymes were also enhanced upon increasing NaCl concentration in the growth medium from 0.5 M to 2.0 M. Under this condition an increaseof approximately 1.5-fold was observed for choline dehydrogenase activity as compared to 2.5-fold for betaine aldehyde dehydrogenase activity, suggesting a preferable induction of the latter enzyme by salt stress. A. halophytica was able to utilize [¹⁴C] ethanolamine and [¹⁴C] glycine for the synthesis of [¹⁴C] glycinebetaine. This revised version was published online in August 2006 with corrections to the Cover Date.     

Homopolygalacturonan Molecular Size in Plant Cell Wall Matrices Via Paramagnetic Ion and Nitroxyl Amide Dipolar Spin-Spin Interactions

Mn²⁺, Cu²⁺, and nitroxyl amines have been shown to bond to plant homopolygalacturonan matrices in a spatially sequential fashion. As a consequence of this special form of cooperativity the lattice constant (κ), determined from Van Vleck's second moment relationship, approaches 1 only when the average number of dipolar interactions per spin approaches 1 (e.g., an array of dimers). Assuming that one paramagnetic ion or nitroxyl amide pair is bonded per polymer block within the matrix when κ = 1, the anionic ligand's average degree of polymerization ([unk]) can be estimated from the concentration of bonded paramagnetic dimers (e.g., [1/χ]_κ~1 = [unk]; χ is the mole fraction of bonded paramagnetic dimers). We have utilized this technique to estimate the average molecular size of homopolygalacturonan blocks in intact higher plant cortical cell walls ([unk] ~83), Nitella cell walls ([unk] ~27) and a commercially available galacturonic acid polymer ([unk] ~35). The [unk] determined from both the intact cortical cell wall lattice and the polygalacturonan were similar to literature values; these findings argue that the electron paramagnetic resonance, (EPR) dipolar spin-spin interaction technique reported herein is a valid approach for estimating molecular size in plant cell walls.     

Alterations in the lipid content of pituitary gland and serum prolactin and growth hormone in cadmium treated rats

The present study was undertaken to assess whether chronic exposition to cadmium (Cd, 0.133 mM per liter for 2 months) through drinking water may affect the lipid contents in the pituitary anterior lobe (PAL) of adult male Wistar rats. As compared to metal non-exposed controls, PALs exposed to cadmium showed an increase in total phospholipid contents, which was associated to an increase of the incorporation of [1–¹⁴C]-methyl choline into phosphatidylcholine and of [U–¹⁴C]-glucose into total phospholipids. The incorporation of [1–¹⁴C]-methyl choline into sphingomyelin was not changed. Incorporation of [1–¹⁴C]-acetate into total fatty acids also increased but incorporation of [1–¹⁴C]-acetate into cholesterol did not change. The activity of phospholipase D decreased both in PALs from Cd exposed rats and in PAL dispersed cells treated with Cd in the culture medium from Cd non-exposed rats. In PALS from Cd exposed rats, a decrease of serum prolactin and growth hormone concentrations was determined. The results shown that cadmium modifies the lipid contents of pituitary gland and directly or indirectly the levels of prolactin and growth hormone in serum.     

Metabolism of gibberellin a(12)-7-aldehyde by soybean cotyledons and its use in identifying gibberellin a(7) as an endogenous gibberellin

The level of gibberellin(GA)-like material in cotyledons of soybean (Glycine max L.) was highest at mid-pod fill—about 10 nanograms GA₃ equivalents per gram fresh weight of tissue, assayed in the immersion dwarf rice bioassay. This amount is about 1000-fold less than levels in Pisum and Phaseolus seed, other legume species whose spectrum of endogenous gibberellins (GAs) is well known. The metabolism of [¹⁴C]-GA₁₂-7-aldehyde (GA₁₂ald)—the universal GA precursor—by intact, mid-pod-fill, soybean cotyledons and their cell-free extracts was investigated. In 4 hours, extracts converted GA₁₂ald to two products—[¹⁴C]GA₁₂ (42% yield) and [¹⁴C]GA₁₅ (7%). Within 5 minutes, intact embryos converted GA₁₂ald to [¹⁴C]GA₁₂ and [¹⁴C]GA₁₅ in 15% yield; 4 hour incubations afforded at least 22 products (96% total yield). The putative [¹⁴C]GA₁₂ was identified as a product of [¹⁴C]GA₁₂ald metabolism on the basis of co-chromatography with authentic GA₁₂ on a series of reversed and normal phase high pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC) systems, and by a dual feed of the putative [¹⁴C]GA₁₂ and authentic [¹⁴C]GA₁₂ to cotyledons of both peas and soybeans. The [¹⁴C]GA₁₅ was identified as a metabolite of [¹⁴C]GA₁₂ald by capillary gas chromatography (GC)-mass-spectrometry-selected ion monitoring, GC-radiocounting, HPLC, and TLC. By adding the [¹⁴C] metabolites of [¹⁴C]GA₁₂ald to a different and larger extract (about 0.2 kg fresh weight of soybean reproductive tissue) and purifying endogenous substances co-chromatographing with these metabolites, at least two GA-like substances were obtained and one identified as GA₇ by GC-mass spectrometry. Since [¹⁴C]GA₉ was not found as a [¹⁴C]metabolite of [¹⁴C]GA₁₂ald, soybean embryos might have a pathway for biosynthesis of active, C-19 gibberellins like that of the cucurbits; GA₁₂ald → GA₁₂ → GA₁₅ → GA₂₄ → GA₃₆ → GA₄ → GA₇.     

Gibberellin metabolism in cell-free extracts from spinach leaves in relation to photoperiod

Cell-free extracts capable of converting [¹⁴C]-labeled gibberellins (GAs) were prepared from spinach (Spinacia oleracea L.) leaves. [¹⁴C]-labeled GAs, prepared enzymically from [¹⁴C]mevalonic acid, were incubated with these extracts, and products were identified by gas chromatography-mass spectrometry. The following pathway was found to operate in extracts from spinach leaves grown under long day (LD) conditions: GA₁₂ → GA₅₃ → GA₄₄ → GA₁₉ → GA₂₀. The pH optima for the enzymic conversions of [¹⁴C]GA₅₃, [¹⁴C]GA₄₄ and [¹⁴C]GA₁₉ were approximately 7.0, 8.0, and 6.5, respectively. These three enzyme activities required Fe²⁺, α-ketoglutarate and O₂ for activity, and ascorbate stimulated the conversion of [¹⁴C]GA₅₃ and [¹⁴C]GA₁₉. Extracts from plants given LD or short days (SD) were examined, and enzymic activities were measured as a function of exposure to LD, as well as to darkness following 8 LD. The results indicate that the activities of the enzymes oxidizing GA₅₃ and GA₁₉ are increased in LD and decreased in SD or darkness, but that the enzyme activity oxidizing GA₄₄ remains high irrespective of light or dark treatment. This photoperiodic control of enzyme activity is not due to the presence of an inhibitor in plants grown in SD. These observations offer an explanation for the higher GA₂₀ content of spinach plants in LD than in SD.     

Evidence for a ferredoxin-dependent choline monooxygenase from spinach chloroplast stroma

Chenopods synthesize betaine in the chloroplast via a two-step oxidation of choline: choline → betaine aldehyde → betaine. Our previous experiments with intact chloroplasts, and in vivo¹⁸O₂ labeling studies, led us to propose that the first step is mediated by a monooxygenase which uses photosynthetically generated reducing power (C Lerma, AD Hanson, D Rhodes [1988] Plant Physiol 88: 695-702). Here, we report the detection of such an activity in vitro. In the presence of O₂ and reduced ferredoxin, the stromal fraction from spinach (Spinacia oleracea) chloroplasts converted choline to betaine aldehyde at rates similar to those in intact chloroplasts (20-50 nanomoles per hour per milligram protein). Incorporation of ¹⁸O from ¹⁸O₂ by the in vitro reaction was demonstrated by fast atom bombardment mass spectrometry. Ferredoxin could be reduced either with thylakoids in the light, or with NADPH plus ferredoxin-NADP reductase in darkness; NADPH alone could not substitute for ferredoxin. No choline-oxidizing activity was detected in the stromal fraction of pea (Pisum sativum L.), a species that does not accumulate betaine. The spinach choline-oxidizing enzyme was stimulated by 10 millimolar Mg²⁺, had a pH optimum close to 8, and was insensitive to carbon monoxide. The specific activity was increased threefold in plants growing in 200 millimolar NaCl. Gel filtration experiments gave a molecular weight of 98 kilodaltons for the choline-oxidizing enzyme, and provided no evidence for other electron carriers which might mediate the reduction of the 98-kilodalton enzyme by ferredoxin.