Biomedical subjects

A D Hanson

Publications and source records attributed to A D Hanson.

At least 55 records · Page 3Linked to original sources

PubMed1988

Choline oxidation by intact spinach chloroplasts.

Plants synthesize betaine by a two-step oxidation of choline (choline --> betaine aldehyde --> betaine). Protoplast-derived chloroplasts of spinach (Spinacia oleracea L.) carry out both reactions, more rapidly in light than in darkness (AD Hanson et al. 1985 Proc Natl Acad Sci USA 82: 3678-3682). We investigated the light-stimulated oxidation of choline, using spinach chloroplasts isolated directly from leaves. The rates of choline oxidation obtained (dark and light rates: 10-50 and 100-300 nanomoles per hour per milligram chlorophyll, respectively) were approximately 20-fold higher than for protoplast-derived chloroplasts. Betaine aldehyde was the main product. Choline oxidation in darkness and light was suppressed by hypoxia. Neither uncouplers nor the Calvin cycle inhibitor glyceraldehyde greatly affected choline oxidation in the light, and maximal choline oxidation was attained far below light saturation of CO(2) fixation. The light stimulation of choline oxidation was abolished by the PSII inhibitors DCMU and dibromothymoquinone, and was partially restored by adding reduced diaminodurene, an electron donor to PSI. Both methyl viologen and phenazine methosulfate prevented choline oxidation. Adding dihydroxyacetone phosphate, which can generate NADPH in organello, doubled the dark rate of choline oxidation. These results indicate that choline oxidation in chloroplasts requires oxygen, and reducing power generated from PSI. Enzymic reactions consistent with these requirements are discussed.

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PubMed1988

Oxygen-18 and deuterium labeling studies of choline oxidation by spinach and sugar beet.

Chenopods synthesize betaine by a two-step oxidation of choline: choline --> betaine aldehyde --> betaine. The pathway is chloroplastic; the first step has been shown in isolated spinach (Spinacia oleracea L.) chloroplasts to be O(2)- and light-dependent, the role of light being to provide reducing power (P Weigel, EA Weretilnyk, AD Hanson 1988 Plant Physiol 86: 54-60). Here, we report use of in vivo(18)O- and (2)H-labeling in conjunction with fast atom bombardment mass spectrometry to test for two hypothetical choline-oxidizing reactions that would explain the observed requirements for O(2) and reductant: a desaturase or an oxygenase. Simple syntheses for (2)H(3)-choline, (2)H(3), (18)O-choline, and (2)H(3), (18)O-betaine are given. A desaturase mechanism was sought by giving choline deuterated at the 2-carbon, or choline unlabeled at this position together with (2)H(2)O and by analyzing newly synthesized betaine. About 15% of the (2)H at C-2 was lost during oxidation of choline to betaine, and about 10% of the betaine made in the presence of 50% (2)H(2)O was monodeuterated. These small effects are more consistent with chemical exchange than with a desaturase, because 10 to 15% losses of (2)H from the C-2 position also occurred if choline was converted to betaine by a purified bacterial choline oxidase. To test for an oxygenase, the incorporation of (18)O from (18)O(2) into newly synthesized betaine was compared with that from (18)O-labeled choline, in light and darkness. Incorporation of (18)O from (18)O-choline was readily detectable and varied from about 15 to 50% of the theoretical maximum value; the (18)O losses were attributable to exchange of the intermediate betaine aldehyde with water. In darkness, incorporation of (18)O from (18)O(2) approached that from (18)O-choline, but in the light was severalfold lower, presumably due to isotopic dilution by photosynthetic (16)O(2). These data indicate that the chloroplast choline-oxidizing enzyme is an oxygenase.

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PubMed1986

Water stress enhances expression of an alpha-amylase gene in barley leaves.

The amylases of the second leaves of barley seedlings (Hordeum vulgare L. cv Betzes) were resolved into eight isozymes by isoelectric focusing, seven of which were beta-amylase and the other, alpha-amylase. The alpha-amylase had the same isoelectric point as one of the gibberellin-induced alpha-amylase isozymes in the aleurone layer. This and other enzyme characteristics indicated that the leaf isozyme corresponded to the type A aleurone alpha-amylase (low pI group). Crossing experiments indicated that leaf and type A aleurone isozymes resulted from expression of the same genes.In unwatered seedlings, leaf alpha-amylase increased as leaf water potential decreased and ABA increased. Water stress had no effect on beta-amylase. alpha-Amylase occurred uniformly along the length of the leaf but beta-amylase was concentrated in the basal half of the leaf. Cell fractionation studies indicated that none of the leaf alpha-amylase occurred inside chloroplasts.Leaf radiolabeling experiments followed by extraction of alpha-amylase by affinity chromatography and immunoprecipitation showed that increase of alpha-amylase activity involved synthesis of the enzyme. However, water stress caused no major change in total protein synthesis. Hybridization of a radiolabeled alpha-amylase-related cDNA clone to size fractionated RNA showed that water-stressed leaves contained much more alpha-amylase mRNA than unstressed plants. The results of these and other studies indicate that regulation of gene expression may be a component in water-stress induced metabolic changes.

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PubMed1986

Induction of lactate dehydrogenase isozymes by oxygen deficit in barley root tissue.

Lactate dehydrogenase (LDH) activity in attached roots of barley and other cereals increased up to 20-fold during several days of severe hypoxia, reaching a maximum of about 2 micromoles per minute per gram fresh weight. In barley, induction of LDH activity was significant at 2.6% O(2) and greatest at 0.06%, the lowest O(2) concentration tested. Upon return to aerobic conditions, induced LDH activity declined with an apparent half-life of 2 days. The isozyme profile of barley LDH comprised 5 bands, consistent with a tetrameric enzyme with subunits encoded by two different Ldh genes. Changes in staining intensity of the isozymes as a function of O(2) level suggested that one Ldh gene was preferentially expressed in severe hypoxia. When tracer [U-(14)C]glucose was supplied to induced roots under hypoxic conditions, lactate acquired label, but much less than either ethanol or alanine. Most of the [(14)C] lactate was secreted into the medium, whereas most other labeled anionic products were retained in the root. Neither hypoxic induction of LDH, nor lactate secretion by induced roots, is predicted from the Davies-Roberts hypothesis, which holds that lactate glycolysis ceases soon after the onset of hypoxia due to acidosis brought about by lactate accumulation in the cytoplasm. These results imply a functional significance for LDH beyond that assigned it in this hypothesis.

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PubMed1986

Purification and properties of hypoxically induced lactate dehydrogenase from barley roots.

Using Affigel Blue and oxamate-agarose affinity chromatography, lactate dehydrogenase (LDH) was purified 2000-fold from hypoxically induced barley roots. Molecular weights of the native and sodium dodecyl sulfate-denatured LDH protein were 157 and 40 kilodaltons, respectively, indicating a tetramer. Purified barley LDH was very similar in size and kinetic properties to potato LDH. However, their amino acid compositions differed substantially and antibodies raised against barley LDH did not cross-react with potato LDH on immunoblots, implying that the barley and potato LDHs are not closely related proteins. In vivo [(35)S] methionine labeling and immunoprecipitation experiments indicated that hypoxia increased the rate of LDH protein synthesis, and immunoblot analysis showed that LDH protein levels rose during hypoxia. We conclude that increased enzyme synthesis plays a major part in the induction of LDH enzyme activity by low O(2) levels in barley roots.

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PubMed1986

Betaine aldehyde oxidation by spinach chloroplasts.

Chenopods synthesize betaine by a two-step oxidation of choline: choline --> betaine aldehyde --> betaine. Both oxidation reactions are carried out by isolated spinach (Spinacia oleracea L.) chloroplasts in darkness and are promoted by light. The mechanism of betaine aldehyde oxidation was investigated with subcellular fractions from spinach leaf protoplasts. The chloroplast stromal fraction contained a specific pyridine nucleotide-dependent betaine aldehyde dehydrogenase (about 150 to 250 nanomoles per milligram chlorophyll per hour) which migrated as one isozyme on native polyacrylamide gels stained for enzyme activity. The cytosol fraction contained a minor isozyme of betaine aldehyde dehydrogenase. Leaves of pea (Pisum sativum L.), a species that lacks betaine, had no betaine aldehyde dehydrogenase isozymes. The specific activity of betaine aldehyde dehydrogenase rose three-fold in spinach plants grown at 300 millimolar NaCl; both isozymes contributed to the increase. Stimulation of betaine aldehyde oxidation in illuminated spinach chloroplasts was due to a thylakoid activity which was sensitive to catalase; this activity occurred in pea as well as spinach, and so appears to be artifactual. We conclude that in vivo, betaine aldehyde is oxidized in both darkness and light by the dehydrogenase isozymes, although some flux via a light-dependent, H(2)O(2)-mediated reaction cannot be ruled out.

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PubMed1985

Betaine synthesis in chenopods: Localization in chloroplasts.

PLANTS FROM SEVERAL FAMILIES (CHENOPODIACEAE, GRAMINEAE, COMPOSITAE) ACCUMULATE BETAINE (GLYCINE BETAINE) IN RESPONSE TO SALT OR WATER STRESS VIA THE PATHWAY: choline --> betainal (betaine aldehyde) --> betaine. Betaine accumulation is probably a metabolic adaptation to stress. Intact protoplasts from leaves of spinach (Spinacia oleracea) oxidized [(14)C]choline to betainal and betaine, as did protoplast lysates. Upon differential centrifugation, the [(14)C]choline-oxidizing activity of lysates sedimented with chloroplasts. Chloroplasts purified from protoplast lysates by a Percoll cushion procedure retained strong [(14)C]choline-oxidizing activity (1-3 nmol/mg of chlorophyll per hr), although the proportion of the intermediate, [(14)C]betainal, in the reaction products was usually higher than for protoplasts. Isolated chloroplasts also readily oxidized [(14)C]betainal to betaine (20-100 nmol/mg of chlorophyll per hr). Light increased the oxidation of both [(14)C]choline and [(14)C]betainal by isolated chloroplasts approximately 3-fold; this light-stimulation was abolished by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Similar results were obtained with another chenopod (Beta vulgaris) but not with pea (Pisum sativum), a species that accumulates no betaine. The chloroplast site for betaine synthesis in chenopods contrasts with the mitochondrial site in mammals.

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PubMed1985

Induction of a Specific N-Methyltransferase Enzyme by Long-Term Heat Stress during Barley Leaf Growth.

Previous work showed that the indole alkaloid gramine accumulates in the upper leaves (e.g. the fifth) of barley as a response to high growth temperatures. The biosynthesis of gramine proceeds from tryptophan to 3-aminomethylindole (AMI); sequential N-methylations of AMI then yield N-methyl-3-aminomethylindole (MAMI) and gramine.To determine whether high-temperature stress increases the activity of gramine pathway enzymes, leaf tissue from plants grown at various temperatures was assayed for N-methyltransferase (NMT) activity using AMI and MAMI as substrates in both in vivo and in vitro assays. NMT activity in expanding fifth leaves was increased 8- to 20-fold by growth at high temperatures (35 degrees C day/30 degrees C night) compared to cool temperatures (15 degrees C/10 degrees C). Several days of high temperature were required for full induction of NMT activity. No induction of NMT activity occurred in leaves which had completed expansion in cool conditions before exposure to high temperature.To investigate NMT induction at the protein level, NMT activity was purified to homogeneity and used to produce polyclonal antibodies. Throughout enzyme purification, relative NMT activities towards AMI and MAMI remained constant, consistent with a single NMT enzyme. Immunoblot analysis showed that a large increase in NMT polypeptide coincided with induction of NMT activity by heat stress. Our results point to a type of high-temperature regulation of gene expression that is quite distinct from heat shock.

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PubMed1984

Three alcohol dehydrogenase genes in wild and cultivated barley: characterization of the products of variant alleles.

Barley (Hordeum vulgare) and its wild progenitor (H. spontaneum) have three loci for alcohol dehydrogenase (EC 1.1.1.1; ADH). The Adh1 locus is constitutively expressed in seed tissues, whereas expression of the loci Adh2 and Adh3 requires anaerobic induction. The Adh3 gene is well expressed in aleurone and embryo tissues kept under N2 for 2-3 days. Using N2-treated embryos, a diverse collection of H. spontaneum was screened in starch gels for electrophoretic variants at the Adh3 locus. Four variants were found: two were conventional mobility variants (Adh3 S, Adh3 V); one was a null variant (Adh3 n); and the fourth (Adh3 I) variant lacked active homodimers and showed reduced heterodimer activity. The 35S-labeled monomers induced under N2 in the lines homozygous for Adh1, Adh2, or Adh3 variants were immunoprecipitated with antiserum raised against maize ADH. Fluorography after separation by SDS-PAGE and by urea-isoelectric focusing indicated that the Adh3 n allele was CRM- and that the Adh3 I gene product was smaller than normal. The Adh1 and Adh3 variants showed independent segregation.

Alcohol Dehydrogenase↗

PubMed1984

Control of lactate dehydrogenase, lactate glycolysis, and alpha-amylase by o(2) deficit in barley aleurone layers.

After 4 days in an atmosphere of N(2), aleurone layers of barley (Hordeum vulgare L. cv Himalaya) remained viable as judged by their ability to produce near normal amounts of alpha-amylases when incubated with gibberellic acid (GA(3)) in air. However, layers did not produce alpha-amylase when GA(3) was supplied under N(2), apparently because alpha-amylase mRNA failed to accumulate.When an 8-hour pulse of [U-(14)C]glucose was supplied under N(2) to freshly prepared aleurone layers, both [(14)C]lactate and [(14)C]ethanol accumulated; the [(14)C]lactate/[(14)C]ethanol ratio was about 0.3. Prior incubation of layers for 1 day under N(2) changed this ratio to about 0.8, indicating an increase in the relative importance of the lactate branch of glycolysis.l(+)Lactate dehydrogenase (LDH) activity was low in freshly prepared aleurone layers and increased 10-fold during 2 days under N(2), whereas alcohol dehydrogenase activity (ADH) was high initially and rose by 60%. The responses of LDH and ADH activities to O(2) tension were dissimilar; when layers were incubated in various O(2)/N(2) mixtures, LDH activity peaked at 2 to 5% O(2) whereas ADH activity was highest at 0% O(2). The LDH activity was resolved into several enzymically active bands by native polyacrylamide gel electrophoresis.We conclude that barley aleurone layers are highly adapted to O(2) deficiency, that they possess an inducible LDH system as well as an ADH system, and we infer that the LDH and ADH systems are independently regulated.

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PubMed1984

Regulated expression of three alcohol dehydrogenase genes in barley aleurone layers.

Three genes specify alcohol dehydrogenase (EC 1.1.1.1.; ADH) enzymes in barley (Hordeum vulgare L.) (Adh 1, Adh 2, and Adh 3). Their polypeptide products (ADH 1, ADH 2, ADH 3) dimerize to give a total of six ADH isozymes which can be resolved by native gel electrophoresis and stained for enzyme activity.Under fully aerobic conditions, aleurone layers of cv Himalaya had a high titer of a single isozyme, the homodimer containing ADH 1 monomers. This isozyme was accumulated by the aleurone tissue during the later part of seed development, and survived seed drying and rehydration. The five other possible ADH isozymes were induced by O(2) deficit. The staining of these five isozymes on electrophoretic gels increased progressively in intensity as O(2) levels were reduced below 5%, and were most intense at 0% O(2).In vivo(35)S labeling and specific immunoprecipitation of ADH peptides, followed by isoelectric focusing of the ADH peptides in the presence of 8 molar urea (urea-IEF) demonstrated the following. (a) Aleurone layers incubated in air synthesized ADH 1 and a trace of ADH 2; immature layers from developing seeds behaved similarly. (b) At 5% O(2), synthesis of ADH 2 increased and ADH 3 appeared. (c) At 2% and 0% O(2), the synthesis of all three ADH peptides increased markedly.Cell-free translation of RNA isolated from aleurone layers, followed by immunoprecipitation and urea-IEF of in vitro synthesized ADH peptides, showed that levels of mRNA for all three ADH peptides rose sharply during 1 day of O(2) deprivation. Northern hybridizations with a maize Adh 2 cDNA clone established that the clone hybridized with barley mRNA comparable in size to maize Adh 2 mRNA, and that the level of this barley mRNA increased 15- to 20-fold after 1 day at 5% or 2% O(2), and about 100-fold after 1 day at 0% O(2).We conclude that in aleurone layers, expression of the three barley Adh genes is maximal in the absence of O(2), that regulation of mRNA level is likely to be a major controlling factor, and that whereas the ADH system of barley has strong similarities to that of maize, it also has some distinctive features.

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PubMed1983

C Tracer Evidence for Synthesis of Choline and Betaine via Phosphoryl Base Intermediates in Salinized Sugarbeet Leaves.

Like other chenopods, sugarbeets (Beta vulgaris L. cv Great Western D-2) accumulate glycine betaine when salinized; this may be an adaptive response to stress. The pathway of betaine synthesis in leaves of salinized (150-200 millimolar NaCl) sugarbeet plants was investigated by supplying [(14)C]formate, phosphoryl[(14)C]monomethylethanolamine ([(14)C][unk] MME) or phosphoryl[(14)C]choline ([(14)C][unk] choline) to leaf discs and following (14)C incorporation into prospective intermediates. The (14)C kinetic data were used to develop a computer model of the betaine pathway.When [(14)C]formate was fed, [unk] MME, phosphoryldimethylethanolamine ([unk] DME) and [unk] choline were the most prominent methylated products at short labeling times, after which (14)C appeared in free choline and in betaine. Phosphatidylcholine labeled more slowly than [unk] choline, choline, and betaine, and behaved as a minor end product. Very little (14)C entered the free methylethanolamines. When [(14)C][unk] MME was supplied, a small amount was hydrolyzed to the free base but the major fate was conversion to [unk] DME, [unk] choline, free choline, and betaine; label also accumulated slowly in phosphatidylcholine. Label from supplied [(14)C][unk] choline entered choline and betaine rapidly, while phosphatidylcholine labeled only slowly and to a small extent.These results are consistent with the pathway [unk] MME -->[unk] DME --> [unk] choline --> choline --> --> betaine, with a minor side branch leading from [unk] choline into phosphatidylcholine. This contrasts markedly (a) with the pathway of stress-induced choline and betaine synthesis in barley, in which phosphatidylcholine apparently acts as an intermediate (Hitz, Rhodes, Hanson 1981, Plant Physiol 68: 814-822); (b) with choline biogenesis in mammalian liver and microorganisms. Computer modeling of the experimental data pointed strongly to regulation at the [unk] choline --> choline step, and also indicated that the rate of [unk] choline synthesis is subject to feedback inhibition by [unk] choline.

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PubMed1983

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 degrees C/16 degrees 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 degrees C/16 degrees C to supra-optimal temperatures (>/=30 degrees C/25 degrees 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 [(14)C]gramine, but neither cultivar degraded it extensively. (b) Arimar leaf tissue incorporated [(14)C]formate label into the N-methyl groups of gramine and MAMI, and converted [methylene-(14)C]tryptophan to AMI, MAMI, and gramine; Proctor leaf tissue did not, even when a trapping pool of unlabeled gramine was supplied. (c) Proctor converted [(14)C]MAMI to gramine as actively as Arimar. (d) Proctor incorporated [(14)C]formate label into gramine and MAMI when supplied with AMI; the ratio [(14)C]gramine/[(14)C]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.

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PubMed1982

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 [(14)C]ethanolamine to young leaf blades or to the taproot sink of intact plants; (b) supplying tracer [(14)C]formate to discs of leaf, hypocotyl, and taproot tissues in darkness. Conversion of both (14)C precursors to betaine was active only in leaf tissue. Very little (14)C 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 (14)C metabolite of both [(14)C]ethanolamine and [(14)C]formate in all tissues of sugar beet. Betaine translocation was examined in young plants of sugar beet and WB-167 by applying tracer [methyl-(14)C]betaine to a young expanded leaf and determining the distribution of (14)C after 3 days. In all cases, extensive (14)C translocation to young leaves and taproot sink occurred; neither in the fed leaf nor in sink organs were any (14)C metabolites of betaine detected.

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PubMed1981

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 [(14)C]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 [(14)C]ethanolamine continued to accumulate in betaine as it was being lost from phosphatidylcholine. When [(14)C]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.The following experiments, in which tracer amounts of [(14)C]ethanolamine or [(14)C]formate were supplied to wilted barley leaves, implicated phosphoryl and phosphatidyl bases as intermediates in the methylation steps between ethanolamine and phosphatidylcholine. Label from both [(14)C]ethanolamine and [(14)C]formate entered phosphorylmonomethylethanolamine and phosphorylcholine very rapidly; these phosphoryl bases were the most heavily labeled products at 15 to 30 minutes after label addition and lost label rapidly as the fed (14)C-labeled precursor was depleted. Phosphatidylmonomethylethanolamine and phosphatidylcholine were also significantly labeled from [(14)C]ethanolamine and [(14)C]formate at early times; the corresponding free bases and nucleotide bases were not. Addition of a trapping pool of phosphorylcholine reduced [(14)C]ethanolamine conversion to both phosphatidylcholine and betaine, and resulted in accumulation of label in the trap.A computer model of the synthesis of betaine via phosphatidylcholine was developed from (14)C kinetic data. The model indicates that about 20% of the total leaf phosphatidylcholine behaves as an intermediate in betaine biosynthesis and that a marked decrease (>/=2-fold) in the half-life of this metabolically active phosphatidylcholine fraction accompanies wilting. Dual labeling experiments with [(14)C]choline and [(3)H]glycerol confirmed that the half-life of the choline portion of phosphatidylcholine falls by a factor of about 2 in wilted leaves.

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PubMed1980

Betaine Synthesis from Radioactive Precursors in Attached, Water-stressed Barley Leaves.

In wilted barley leaves, betaine accumulates at about 200 nanomoles per 10 centimeters leaf per day. Results with (14)C-labeled precursors were qualitatively and quantitatively consistent with de novo synthesis of this betaine from serine via ethanolamine, choline, and betaine aldehyde and indicated that water stress may increase the activities of all steps in this pathway except the last.Doses (1 micromole) of each (14)C-labeled precursor were supplied as droplets to the tips of attached, 10-centimeter, second-leaf blades of turgid and wilted plants, and the incorporation of (14)C into betaine was followed. From the rates of betaine labeling, estimates were made of the potential capacities (nanomoles per 10 centimeters leaf per day) for the methylation and oxidation steps. Labeling of betaine from [(14)C]choline, [(14)C]ethanolamine, and [(14)C]serine was about 7- to 10-fold greater in leaves wilted for 2 days than in turgid leaves, whereas label from [(14)C]betaine aldehyde appeared in betaine at about the same rate in both turgid and wilted leaves. In leaves wilted for 2 days, the potential capacities for converting [(14)C]ethanolamine, [(14)C]choline, and [(14)C]betaine aldehyde to betaine all approached or exceeded the rate of betaine accumulation (about 200 nanomoles per 10 centimeters leaf per day); in turgid leaves, only the potential for converting betaine aldehyde to betaine exceeded this rate. The rate of conversion of [(14)C]ethanolamine to betaine increased 4-fold after 6 to 10 hours of wilting, which was soon enough to account for the onset of betaine accumulation.

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PubMed1979

Proline Accumulation in Water-stressed Barley Leaves in Relation to Translocation and the Nitrogen Budget.

Mobilization of N from leaves of barley (Hordeum vulgare L.) during water stress, and the role of proline as a mobilized species, were examined in plants at the three-leaf stage. The plants responded to water stress by withdrawing about 25% of the total reduced N from the leaf blades via phloem translocation. Most of this N loss was during the first 2 days while translocation of (14)C-photosynthate out of the stressed blade still remained active. Free proline accumulation in the blade was initially slow, and became more rapid during the 2nd day of stress. Although a major free amino acid, proline accounted for only about 5% of the total N (soluble + insoluble) retained in severely stressed blades. When the translocation pathway in water-stressed leaves was interrupted just below the blade by a heat girdle, a cold jacket, or by blade excision, N loss from the blade was prevented and proline began to accumulate rapidly on 1st day of stress. Little free proline accumulated in the blades until after the ability to translocate was lost. Proline was, however, probably not a major species of N translocated during stress, because proline N accumulation in heat-girdled stressed leaves was five times slower than the rate of total N export from intact blades.

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PubMed1979

Amino Acids Translocated from Turgid and Water-stressed Barley Leaves: I. Phloem Exudation Studies.

The phloem exudation technique of King and Zeevaart (Plant Physiol 1974 53: 96-103) was modified for use with barley plants, to investigate the effect of water stress upon amino acid translocation at seedling and grainfilled stages.Seedling leaves and flag leaves from unstressed and moderately water-stressed plants exuded (14)CO(2) assimilates, sugars, and amino acids when their sheaths were cut and immersed in a 5 millimolar solution of Na(2)EDTA (pH 7.0). By including PEG 6000 (-10 bars) in the Na(2)EDTA solution, leaves severed from moderately water-stressed plants could be maintained in a wilted state. Such leaves produced about as much exudate as turgid leaves of unstressed plants.The following observations suggest a phloem origin for most of the exudate. Exudation was markedly stimulated by light and by CO(2) enrichment. The release of NO(3) (-) declined after cutting, and did not parallel exudation of (14)CO(2) assimilates, sugar, and amino acids. The relative quantities and specific radioactivities of sugars and amino acids in the exudate differed from those of sugars and amino acids extracted from sheath tissue.Major amino acids in exudate from unstressed seedling and flag leaves were glutamine, glutamate, serine, alanine, and aspartate; proline was virtually absent. Exudate from water-stressed leaves contained relatively more serine, and also some proline and gamma-aminobutyric acid.

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