Phosphoethanolamine bases as intermediates in phosphatidylcholine synthesis by lemna

The pathway for synthesis of phosphatidylcholine, the dominant methyl-containing end product formed by Lemna paucicostata, has been investigated. Methyl groups originating in methionine are rapidly utilized by intact plants to methylate phosphoethanolamine successively to the mono-, di-, and tri-methyl (i.e. phosphocholine) phosphoethanolamine derivatives. With continued labeling, radioactivity initially builds up in these compounds, then passes on, accumulating chiefly in phosphatidylcholine (34% of the total radioactivity taken up by plants labeled to isotopic equilibrium with l-[(14)CH(3)]methionine), and in lesser amounts in soluble choline (6%). Radioactivity was detected in mono- and dimethyl derivatives of free ethanolamine or phosphatidylethanolamine only in trace amounts. Pulse-chase experiments with [(14)CH(3)]choline and [(3)H] ethanolamine confirmed that phosphoethanolamine is rapidly methylated and that phosphocholine is converted to phosphatidylcholine. Initial rates indicate that methylation of phosphoethanolamine predominates over methylation of either phosphatidylethanolamine or free ethanolamine at least 99:1. Although more studies are needed, it is suggested this pathway may well turn out to account for most phosphatidylcholine synthesis in higher plants. Phosphomethylethanolamine and phosphodimethylethanolamine are present in low quantities during steady-state growth (18% and 6%, respectively, of the amount of phosphocholine). Radioactivity was not detected in CDP-choline, probably due to the low steady-state concentration of this nucleotide.     

Phosphatidylcholine synthesis: differing patterns in soybean and carrot

The methylation steps in the biosynthesis of phosphatidylcholine by tissue culture preparations of carrot (Daucus carota L.) and soybean (Glycine max), and by soybean leaf discs, have been studied. Preparations were incubated with tracer concentrations of l-[³H₃C]methionine and the kinetics of appearance of radioactivity in phosphomethylethanolamine, phosphodimethylethanolamine, phosphocholine, phosphatidylmethylethanolamine, phosphatidyldimethylethanolamine, phosphatidylcholine, methylethanolamine, dimethylethanolamine, and choline followed at short incubation times. With soybean (tissue culture or leaves), an initial methylation utilizes phosphoethanolamine as substrate, forming phosphomethylethanolamine. The latter is converted to phosphatidylmethylethanolamine, which is successively methylated to phosphatidyldimethyethanolamine and to phosphatidylcholine. With carrot, again, an initial methylation is of phosphoethanolamine. Subsequent methylations occur at both the phospho-base and phosphatidyl-base levels. Both of these patterns differ qualitatively from that previously demonstrated in Lemna (SH Mudd, AH Datko 1986 Plant Physiol 82: 126-135) in which all three methylations occur at the phospho-base level. For soybean and carrot, some added contribution from initial methylation of phosphatidylethanolamine has not been excluded. These results, together with those from similar experiments carried out with water-stressed barley leaves (WD Hitz, D Rhodes, AD Hanson 1981 Plant Physiol 68: 814-822) and salinized sugarbeet leaves (AD Hanson, D Rhodes 1983 Plant Physiol 71: 692-700) suggest that in higher plants some, perhaps all, phosphatidylcholine synthesis occurs via a common committing step (conversion of phosphoethanolamine to phosphomethylethanolamine) followed by a methylation pattern which differs from plant to plant.     

Enzymes of ureide synthesis in pea and soybean

Soybean (Glycine max) and pea (Pisum sativum) differ in the transport of fixed nitrogen from nodules to shoots. The dominant nitrogen transport compounds for soybean are ureides, while amides dominate in pea. A possible enzymic basis for this difference was examined.

The level of enzymes involved in the formation of the ureides allantoin and allantoic acid from inosine 5′-monophosphate (IMP) was compared in different tissues of pea and soybean. Two enzymes, 5′-nucleotidase and uricase, from soybean nodules were found to be 50- and 25-fold higher, respectively, than the level found in pea nodules. Other purine catabolizing enzymes (purine nucleosidase, xanthine dehydrogenase, and allantoinase) were found to be at the same level in the two species. From comparison of enzyme activities in nodules with those from roots, stems, and leaves, two enzymes were found to be nodule specific, namely uricase and xanthine dehydrogenase. The level of enzymes found in the bacteroids indicated no significant contribution of Rhizobium japonicum purine catabolism in the overall formation of ureides in the soybean nodule. The presence in the nodules of purine nucleosidase and ribokinase activities makes a recirculation of the ribose moiety possible. In concert with phosphoribosylpyrophosphate synthetase, ribose becomes available for a new round of purine de novo synthesis, and thereby ureide formation.

     

Enzymes of purine catabolism in soybean plants

Remarkable formation and utilization of allantoin is observedin soybean (Glycine max variety A62-1). To study this, variousenzymes involved in purine catabolism (i.e., xanthine oxidase,uricase and allantoinase) were measured in different regionsof soybean plants during development. Uricase, which catalyzesthe direct formation of allantoin from uric acid, was studiedin detail. The activities of these three enzymes were highest in the rootnodules, indicating that the nodules are the major site of allantoinmetabolism. Radicles only showed appreciable activity about80 hr after the seeds were planted. Allantoinase activity wasdetected in all regions tested, showing that allantoin translocatedfrom the nodules can be metabolized in the roots, stem and leaves.In the nodules, xanthine oxidase was localized in the nuclearfraction, while uricase was mainly restricted to the mitochondrialfraction and allantoinase to the soluble fraction. Uricase was partially purified from the nodules and radicles,respectively. The pH optimum of enzyme from the nodules was9.5, whereas that of enzyme from the radicles was 7.0. The enzymefrom the nodules did not require a cofactor, while that fromthe radicles showed an absolute requirement for a cofactor,which was a low molecular substance easily separable from theapoprotein. Thus, the uricase in nodules differs in chemicalproperties from that in the host plant. The results are discussedin relation to change in the allantoin level in soybean tissues. (Received November 1, 1974; )     

Regulation of lysine and threonine synthesis in carrot cell suspension cultures and whole carrot roots

Aspartokinase (EC 2.7.2.4), homoserine-dehydrogenase (EC 1.1.1.3) and dihydrodipicolinic-acid-synthase (EC 4.2.1.52) activities were examined in extracts from 1-year-old and 11-year-old cell suspension cultures and whole roots of garden carrot (Daucus carota L.). Aspartokinase activity from suspension cultures was inhibited 85% by 10 mM L-lysine and 15% by 10mM L-threonine. In contrast, aspartokinase activity from whole roots was inhibited 45% by 10 mM lysine and 55% by 10 mM threonine. This difference may be based upon alterations in the ratios of the two forms (lysine-and threonine-sensitive) of aspartokinase, since the activity is consistently inhibited 100% by lysine+threonine. Only one form each of homoserine dehydrogenase and of dihydrodipicolinic acid synthase was found in extracts from cell suspension cultures and whole roots. The regulatory properties of either enzyme were identical from the two sources. In both the direction of homoserine formation and aspartic--semialdehyde formation, homoserine dehydrogenase activities were inhibited by 10mM threonine and 10 mM L-cysteine in the presence of NADH or NADPH. KCl increased homoserine dehydrogenase activity to 185% of control values and increased the inhibitory effect of threonine. Dihydrodipicolinic acid synthase activities from both sources were inhibited over 80% by 0.5 mM lysine. Aspartokinase was less sensitive to inhibition by low concentrations of lysine and threonine than were dihydrodipicolinic acid synthase and homoserine dehydrogenase to inhibition by the respective inhibitors.     

Yeast mutant defective in phosphatidylcholine synthesis

The Saccharomyces cerevisiae opi3-3 mutant was shown to be defective in the synthesis of phosphatidylcholine via methylation of phosphatidylethanolamine. The opi3-3 mutant was isolated on the basis of an inositol excretion phenotype and was not auxotrophic for choline. Inositol, but not choline, stimulated growth of the mutant. The opi3-3 mutation was recessive and was genetically linked to the ino4 locus. When grown in the absence of exogenous choline, the opi3-3 mutant had a phospholipid composition consisting of 2 to 3% phosphatidylcholine compared with 40 to 50% in wild-type strains. In addition, the mutant accumulated elevated amounts of two intermediates, phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine. The incorporation of label from [methyl-14C]methionine into phosphatidylcholine was reduced 80 to 90% in the mutant compared with wild-type strains. However, label was recovered in the intermediates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine. The mutant is believed to be defective in the third and possibly the second methylation reaction in the formation of phosphatidylcholine from phosphatidylethanolamine. The first methylation reaction appeared to be occurring at normal or even elevated levels. Based upon incorporation of choline into phosphatidylcholine, it is concluded that the opi3-3 mutant has no defect in the synthesis of phosphatidylcholine from exogenous choline. Furthermore, phosphatidylcholine represents over 25% of the phospholipid composition of the mutant when it is grown in the presence of exogenous choline.     

Enzymes of amide and ureide biogenesis in developing soybean nodules

Amide and ureide biogenic enzymes were measured in the plant fraction of soybean (Glycine max) nodules during the period 11 to 23 days after inoculation with Rhizobium japonicum (USDA 3I1b142). Enzymes involved in the initial assimilation of ammonia, i.e. glutamine synthetase, glutamate synthase, and aspartate aminotransferase, showed substantial increases in their specific activities over the time course. These increases paralleled the induction of nitrogenase activity in the bacteroid and leghemoglobin synthesis in the plant fraction. The specific activity of asparagine synthetase, however, showed a rapid decline after an initial increase in specific activity. Following the initial increases in the ammonia assimilatory enzymes, there was an increase in the activity of 5-phosphoribosylpyrophosphate amidotransferase, the enzyme which catalyzes the first committed step of de novo purine biosynthesis. This was followed by a dramatic increase in the purine oxidative enzymes, xanthine dehydrogenase and uricase. Smaller increases were observed in the activities of enzymes associated with the supply of metabolites to the purine biosynthetic pathway: phosphoglycerate dehydrogenase, serine hydroxymethylase, and methylene tetrahydrofolate dehydrogenase.     

Reversal of the effects of a night interruption in lemna by inhibitors of ribonucleic Acid synthesis

The inhibition of flowering of Lemna perpusilla Torr. strain 6746 caused by a light break can be partially reversed by treatment with actinomycin D or 2-thiouracil. Actinomycin D is most active in reversing the response to a light break if the inhibitor is present in the fronds at the time the light break is administered.     

Synthesis of methylated ethanolamine moieties: regulation by choline in soybean and carrot

The results of experiments in which intact plants of Lemna paucicostata were labeled with either l-[³H₃C]methionine, l-[¹⁴CH₃]methionine, or [1,2-¹⁴C]ethanolamine support the conclusion that growth in concentrations of choline of 3.0 micromolar or above brings about marked decreases in the rate of biosynthesis of methylated forms of ethanolamine (normally present chiefly as phosphatidylcholine, with lesser amounts of choline and phosphocholine). The in vivo locus of the block is at the committing step in the biosynthetic sequence at which phosphoethanolamine is methylated by S-adenosylmethionine to form phosphomethylethanolamine. The block is highly specific: flow of methyl groups originating in methionine continues into S-adenosylmethionine, S-methylmethionine, the methyl moieties of pectin methyl ester, and other methylated metabolites. When choline uptake is less than the total that would be synthesized by control plants, phosphoethanolamine methylation is down-regulated to balance the uptake; total plant content of choline and its derivatives remains essentially constant. At maximum down-regulation, phosphoethanolamine methylation continues at 5 to 10% of normal. A specific decrease in the total available activity of AdoMet: phosphoethanolamine N-methyltransferase, as well as feedback inhibition of this enzyme by phosphocholine, and prevention of accumulation of phosphoethanolamine by down-regulation of ethanolamine synthesis may each contribute to effective control of phosphoethanolamine methylation. This down-regulation may necessitate major changes in S-adenosylmethionine metabolism. Such changes are discussed.     

Sucrose suppression of chlorophyll synthesis in carrot callus cultures

Summary Substrate levels of sucrose were shown to reduce chlorophyll synthesis in carrot tissue culture strain CRT1 but not in strain CRT2. In CRT1 the effect was shown to be a suppression of greening specifically by sucrose rather than a reducing sugar requirement for chlorophyll synthesis. In CRT1 sucrose caused both a reduction in chloroplast numbers per cell and a suppression of lamellar development in plastids. This effect on chloroplast structure was consistent with the observed reduced photosynthetic efficiency (micromoles CO₂ per hour per mg chlorophyll) of CRT1 calluses grown on sucrose.     

Carotenoid synthesis in a suspension culture of carrot cells

Biosynthesis of carotenoid in cultured carrot cells was studiedin relation to cell growth and acetate metabolism. Of the twostrains tested, one (GD-1) predominantly produces ß-caroteneand the other (GD-2) lycopene. In both strains, carotenoid wasproduced in parallel with cell growth. Incorporations of acetate-¹⁴Cinto carotenoids, organic acids and amino acids were acceleratedby increasing the concentration of 2,4-D in the medium. (Received November 17, 1970; )     

Enzymes of sucrose breakdown in soybean nodules: alkaline invertase

The specific activities of acid and alkaline invertases (β-d-fructofuranoside fructohydrolase, EC 3.2.1.26), sucrose synthase (UDPglucose: d-fructose 2-α-d-glucosyltransferase, EC 2.4.1.13), hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1), and fructokinase (ATP: d-fructose 6-phosphotransferase, EC 2.7.1.4) were determined in soybean (Glycine max L. Merr cv Williams) nodules at different stages of development and, for comparison, in roots of nonnodulated soybeans. Alkaline invertase and sucrose synthase were both involved in sucrose metabolism in the nodules, but there was only a small amount of acid invertase present. The nodules contained more phosphorylating activity with fructose than glucose. Essentially all of the alkaline invertase, sucrose synthase, and fructokinase were in the soluble fraction of nodule extracts whereas hexokinase was in the bacteroid, plant particulate, and soluble fractions.     

Relationships between phosphatidylcholine content, chitin synthesis, growth, and morphology of Aspergillus nidulans choC

The phosphatidylcholine (PC) content of Aspergillus nidulans choC was varied by growing the auxotroph in medium containing various concentrations of choline chloride. Direct linear correlations were observed between PC content and in vivo chitin synthase activity, between in vivo chitin synthase activity and mean hyphal extension rate, and between mean hyphal extension rate and hyphal growth unit length; hyphal growth unit length is a measure of hyphal branching. Further, there was a correlation between PC content and colony radial growth rate. Thus, membrane composition is an important determinant of both hyphal (and colony) extension rate and mycelial morphology.     

In vivo metabolism of 5'-methylthioadenosine in lemna

Evidence is presented that Lemna converts 5′-methylthioadenosine (MTA) to methionine. The methylthio moiety and four of the ribose carbons of the nucleoside contribute the methylthio and the four-carbon moieties of methionine. Plants grown in the presence of inhibitors which block methionine biosynthesis convert MTA to methionine at a rate sufficient to sustain normal growth (at least 4.4 nanomoles per colony per doubling with a molar yield of at least 65%). The pathway for conversion is shown to be constitutive in plants grown in standard medium and to function at a rate sufficient to dispose of MTA arising as a result of polyamine synthesis, and to explain the observed rate (1.4 nanomoles per colony per doubling) of preferential recycling of methionine sulfur (Giovanelli, Mudd, Datko 1981 Biochem Biophys Res Commun 100: 831-839). Rapid entry of methionine methyl into S-adenosylmethionine and phosphorylcholine was observed for plants grown in standard medium. Adenine generated during this cycle is efficiently salvaged into ADP and ATP.

Conversion of MTA to methionine completes the steps in methionine thiomethyl recycling (Giovanelli, Mudd, Datko 1981 Biochem Biophys Res Commun 100: 831-839) in which the sulfur of methionine is retained while the four-carbon moiety is not. The findings further show that the four-carbon moiety of methionine can be derived via the ribose moiety of MTA in addition to the established route from O-phosphohomoserine via transsulfuration. Previous observations (Giovanelli, Mudd, Datko 1980 Biochemistry of Plants pp 453-505) can now be interpreted as establishing that exogenous methionine down-regulates its own net synthesis via the transsulfuration pathway.

     

Effect of propylthiouracil-induced hypothyroidism on phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin synthesis in chick liver microsomes.

Biosynthesis of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin was studied in liver endoplasmic reticulum obtained from newly hatched chicks which were made hypothyroid by feeding 0.2% propylthiouracil. In vitro measurements were made of the specific activities of phosphorylcholine-glyceride (cholinephosphotransferase (EC 2.7.8.2), hosphorylethanolamine-glyceride (ethanolamine-phosphotransferase (EC 2.7.8.1)), and phosphorylcholine-ceramide (ceramide cholinephosphotransferase (EC 2.7.8.3)) transferases in control and hypothyroid chick liver for a period of 40 days. The specific activity of all three transferases began to decline after the chicks were on the propylthiouracil-containing diet for 5 days and steadily declined, reaching levels 10-15% of the controls after 15 days. These low levels were maintained for as long as the chicks were on this diet. Administration of L-thyroxine (15 mug/100 g of body weight) to the hypothyroid chicks caused a marked increase in the specific activities of all three transferases, reaching levels similar to those seen in the control chicks in 36-48 h. The specific activities then declined as the chicks were maintained on the diet of propylthiouracil, reaching the former low levels after 120 h. Administration of cycloheximide alone to the hypothyroid chicks caused a rise in the specific activities of the transferases after 24 h approximately equal to that caused by thyroxine alone, while thyroxine and cycloheximide together were no different than either alone. These studies indicate that in some manner circulating thyroxine controls the activities of enzymes involved in the biosynthesis of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin in chick liver endoplasmic reticulum. There was no evidence that induction of hypothyroidism by propylthiouracil had any effect on the activities of these enzymes in the CNS.     

Enzymes of asparagine synthesis in maize roots

An asparagine synthetase which is active with either glutamine or NH ₄ ⁺ has been found in maize (Zea mays L.) roots. Unlike the enzyme obtained from legume cotyledons, the maize-root enzyme is only slightly more efficient with glutamine (Km, 1.0 mM) than with NH ₄ ⁺ (Km, 2.0–3.0 mM). The activity of this enzyme is higher in the mature root than in the root-tip region, i.e. root cells develop a capacity to make asparagine from glutamine or NH ₄ ⁺ as they mature. -Cyanoalanine synthetase is also present in maize roots. The apparent Km for cysteine is 2.6 mM and for cyanide is 0.57 mM. The enzyme is more active in the root tip than in mature root tissue. Thus, if asparagine were made in the root tip, the cyanide pathway could represent the mechanism of synthesis. It is our contention, however, that this potential is not realized under normal conditions because ¹⁴C-experiments performed previously have indicated a limited availability of both CN and cysteine in the maize root.     

Enzymes of triacylglycerol synthesis and their regulation

Since the pathways of glycerolipid biosynthesis were elucidated in the 1950's, considerable knowledge has been gained about the enzymes that catalyze the lipid biosynthetic reactions and the factors that regulate triacylglycerol biosynthesis. In the last few decades, in part due to advances in technology and the wide availability of nucleotide and amino acid sequences, we have made enormous strides in our understanding of these enzymes at the molecular level. In many cases, sequence information obtained from lipid biosynthetic enzymes of prokaryotes and yeast has provided the means to search the genomic and expressed sequence tag databases for mammalian homologs and most of the genes have now been identified. Surprisingly, multiple isoforms appear to catalyze the same chemical reactions, suggesting that each isoform may play a distinct functional role in the pathway of triacylglycerol and phospholipid biosynthesis. This review focuses on the de novo biosynthesis of triacylglycerol in eukaryotic cells, the isoenzymes that are involved, their subcellular locations, how they are regulated, and their putative individual roles in glycerolipid biosynthesis.