Betaine Synthesis Research Articles

Effect of Soil Salinity on the Expression of Betaine Aldehyde Dehydrogenase in Leaves: Investigation of Hydraulic, Ionic and Biochemical Signals.

Betaine aldehyde dehydrogenase (BADH) catalyses the last step in glycine betaine synthesis. The levels of BADH enzyme and BADH mRNA have previously been shown to be increased several-fold by salt stress. To characterise this induction more thoroughly, BADH mRNA levels and enzyme activities were analysed in leaves of sugar beet plants (Beta vulgaris L.) subjected to different salinisation regimes. Following a salt shock (transfer from 0 to 400 mM NaCI) BADH enzyme activity rose slowly for several days. In contrast, BADH mRNA level first decreased for several hours, and then increased. When salt was leached from the rooting medium of salinised plants, BADH enzyme activity declined, with a half-life of more than 4 days. However, the level of BADH mRNA declined sharply with an apparent half-life of 2 h showing that transcription of the BADH gene or the stability of BADH mRNA in leaves can respond very dynamically to salinity changes around the root. In plants which had been gradually salinised and then held at various NaCl concentrations, the steady state level of enzyme rose continuously between 0 and 500 mM NaCl, whereas that of BADH mRNA reached a plateau at 100 mM NaCl. In general, the observed BADH mRNA fluctuations could not be satisfactorily explained by assuming them to be responses to hydraulic signals. This suggests the participation of a non-hydraulic signal or signals coming from the root. The non-hydraulic signal is unlikely to be NaCl, because leaf disks exposed to salt concentrations typical of the apoplast of salinised leaves did not accumulate BADH mRNA. A biochemical messenger is thus implied. Although abscisic acid application to leaf disks elicited significant increases in BADH mRNA level, these were several-fold smaller than those observed in leaves of intact salinised plants, suggesting the involvement of some other substance.

Comparative Physiological Evidence that β-Alanine Betaine and Choline-O-Sulfate Act as Compatible Osmolytes in Halophytic Limonium Species

The quaternary ammonium compounds accumulated in saline conditions by five salt-tolerant species of Limonium (Plumbaginaceae) were analyzed by fast atom bombardment mass spectrometry. Three species accumulated beta-alanine betaine and choline-O-sulfate; the others accumulated glycine betaine and choline-O-sulfate. Three lines of evidence indicated that beta-alanine betaine and choline-O-sulfate replace glycine betaine as osmo-regulatory solutes. First, tests with bacteria showed that beta-alanine betaine and choline-O-sulfate have osmoprotective properties comparable to glycine betaine. Second, when beta-alanine betaine and glycine betaine accumulators were salinized, the levels of their respective betaines, plus that of choline-O-sulfate, were closely correlated with leaf solute potential. Third, substitution of sulfate for chloride salinity caused an increase in the level of choline-O-sulfate and a matching decrease in glycine betaine level. Experiments with (14)C-labeled precursors established that beta-alanine betaine accumulators did not synthesize glycine betaine and vice versa. These experiments also showed that beta-alanine betaine synthesis occurs in roots as well as leaves of beta-alanine betaine accumulators and that choline-O-sulfate and glycine betaine share choline as a precursor. Unlike glycine betaine, beta-alanine betaine synthesis cannot interfere with conjugation of sulfate to choline by competing for choline and does not require oxygen. These features of beta-alanine betaine may be advantageous in sulfate-rich salt marsh environments.

Distribution of compatible solutes in the halophilic methanogenic archaebacteria

Accumulation of compatible solutes, by uptake or de novo synthesis, enables bacteria to reduce the difference between osmotic potentials of the cell cytoplasm and the extracellular environment. To examine this process in the halophilic and halotolerant methanogenic archaebacteria, 14 strains were tested for the accumulation of compatible solutes in response to growth in various extracellular concentrations of NaCl. In external NaCl concentrations of 0.7 to 3.4 M, the halophilic methanogens accumulated K+ ion and low-molecular-weight organic compounds. beta-Glutamate was detected in two halotolerant strains that grew below 1.5 M NaCl. Two unusual beta-amino acids, N epsilon-acetyl-beta-lysine and beta-glutamine (3-aminoglutaramic acid), as well as L-alpha-glutamate were compatible solutes among all of these strains. De novo synthesis of glycine betaine was also detected in several strains of moderately and extremely halophilic methanogens. The zwitterionic compounds (beta-glutamine, N epsilon-acetyl-beta-lysine, and glycine betaine) and potassium were the predominant compatible solutes among the moderately and extremely halophilic methanogens. This is the first report of beta-glutamine as a compatible solute and de novo biosynthesis of glycine betaine in the methanogenic archaebacteria.

Betaine Deficiency in Maize

Maize (Zea mays L.) is a betaine-accumulating species, but certain maize genotypes lack betaine almost completely; a single recessive gene has been implicated as the cause of this deficiency (D Rhodes, PJ Rich [1988] Plant Physiol 88: 102-108). This study was undertaken to determine whether betaine deficiency in diverse maize germplasm is conditioned by the same genetic locus, and to define the biochemical lesion(s) involved. Complementation tests indicated that all 13 deficient genotypes tested shared a common locus. One maize population (P77) was found to be segregating for betaine deficiency, and true breeding individuals were used to produce related lines with and without betaine. Leaf tissue of both betaine-positive and betaine-deficient lines readily converted supplied betaine aldehyde to betaine, but only the betaine-containing line was able to oxidize supplied choline to betaine. This locates the lesion in betaine-deficient plants at the choline --> betaine aldehyde step of betaine synthesis. Consistent with this location, betaine-deficient plants were shown to have no detectable endogenous pool of betaine aldehyde.

Molecular cloning of a plant betaine-aldehyde dehydrogenase, an enzyme implicated in adaptation to salinity and drought.

Many plants, as well as other organisms, accumulate betaine (N,N,N-trimethylglycine) as a nontoxic or protective osmolyte under saline or dry conditions. In plants, the last step in betaine synthesis is catalyzed by betaine-aldehyde dehydrogenase (BADH, EC 1.2.1.8), a nuclear-encoded chloroplastic enzyme. A cDNA clone for BADH (1812 base pairs) was selected from a lambda gt10 cDNA library derived from leaves of salt-stressed spinach (Spinacia oleracea L.). The library was screened with oligonucleotide probes corresponding to amino acid sequences of two peptides prepared from purified BADH. The authenticity of the clone was confirmed by nucleotide sequence analysis; this analysis demonstrated the presence of a 1491-base-pair open reading frame that contained sequences encoding 12 peptide fragments of BADH. The clone hybridized to a 1.9-kilobase mRNA from spinach leaves; this mRNA was more abundant in salt-stressed plants, consistent with the known salt induction of BADH activity. The amino acid sequence deduced from the BADH cDNA sequence showed substantial similarities to those for nonspecific aldehyde dehydrogenases (EC 1.2.1.3 and EC 1.2.1.5) from several sources, including absolute conservation of a decapeptide in the probable active site. Comparison of deduced and determined amino acid sequences indicated that the transit peptide may comprise only 7 or 8 residues, which is atypically short for precursors to stromal proteins.

Comparative biochemical and immunological studies of the glycine betaine synthesis pathway in diverse families of dicotyledons

Members of the Chenopodiaceae can accumulate high levels (>100 μmol·(g DW)(-1)) of glycine betaine (betaine) in leaves when salinized. Chenopodiaceae synthesize betaine by a two-step oxidation of choline (choline→betaine aldehyde→ betaine), with the second step catalyzed by betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8). High betaine levels have also been reported in leaves of species from several distantly-related families of dicotyledons, raising the question of whether the same betaine-synthesis pathway is used in all cases.Fast atom bombardment mass spectrometry showed that betaine levels of >100 μmol·(g DW)(-1) are present in Lycium ferocissimum Miers (Solanaceae), Helianthus annuus L. (Asteraceae), Convolvulus arvensis L. (Convolvulaceae), and Amaranthus caudatus L. (Amaranthaceae), that salinization promotes betaine accumulation in these plants, and that they can convert supplied choline to betaine aldehyde and betaine. Nicotiana tabacum L. and Lycopersicon lycopersicum (L.) Karst. ex Farw. (Solanaceae), Lactuca sativa L. (Asteraceae) and Ipomoea purpurea L. (Convolvulaceae) also contained betaine, but at a low level (0.1-0.5 μmol·(g DW)(-1). Betaine aldehyde dehydrogenase activity assays, immunotitration and immunoblotting demonstrated that the betaine-accumulating species have a BADH enzyme recognized by antibodies raised against BADH from Spinacia oleracea L. (Chenopodiaceae), and that the Mr of the BADH monomer is in all cases close to 63 000. These data indicate that the choline→betaine aldehyde→betaine pathway may have evolved by vertical descent from an early angiosperm ancestor, and might be widespread (albeit not always strongly expressed) among flowering plants. Consistent with these suggestions, Magnolia x soulangiana was found to have a low level of betaine, and to express a protein of Mr 63 000 which cross-reacted with antibodies to BADH from Spinacia oleracea.

CO2 Fixation Rate and RuBisCO Content Increase in the Halotolerant Cyanobacterium, Aphanothece halophytica, Grown in High Salinities

The growth of the halotolerant cyanobacterium Aphanothece halophytica, previously adapted to 0.5 molar NaCl, was optimal when NaCl concentration in culture medium was in the range 0.5 to 1.0 molar. The growth was delayed at either too low or too high salinities with lag time of ca. 0.5 day in 0.25 molar NaCl and ca. 2 days in 2 molar NaCl under the experimental conditions. However, the growth rates at the logarithmic phase were similar in the culture media containing NaCl in the range 0.25 to 2.0 molar. The capacity of photosynthetic CO(2) fixation increased 3.7-fold in the cells at the logarithmic phase as NaCl concentration in the culture medium increased from 0.25 to 2.0 molar. The protein level of ribulose 1,5-bisphosphate carboxylase/oxygenase was also found to increase with increasing salinity using both an immunoblotting method and protein A-gold immunoelectron microscopy. These results indicate that high photosynthetic capacity and high ribulose 1,5-bisphosphate carboxylase/oxygenase content may entail an important role in betaine synthesis and adaptation of the A. halophytica cells to high NaCl level.

Lac fusion analysis of the bet genes of Escherichia coli: regulation by osmolarity, temperature, oxygen, choline, and glycine betaine.

The synthesis of glycine betaine, a powerful osmoprotectant, from its precursor, choline, is a function of the bet genes. The bet genes code for the high-affinity transport of choline and the enzymes for its conversion to glycine betaine. These genes map at 7.5 min on the E. coli chromosome and are contained on the conjugative plasmid F'2. To study the transcriptional regulation of the bet genes in response to various environmental conditions, a collection of 30 lac operon fusions was isolated by utilizing the bet genes contained on F'2. Four osmoregulated bet loci (betA, betB, betC, and betT) were identified based on biochemical, regulatory, and merodiploid analysis of these fusions. All of the bet fusions demonstrated a 7- to 10-fold increase in transcription in response to increases in the osmotic strength of the growth medium. Choline further induced expression of lac fusions at the betA, betB, and betT loci when the cells were grown under conditions of osmotic stress. The end product of the pathway, glycine betaine, was a corepressor of choline induction for fusions at the betA and betT loci. Expression of the betA, betB, and betT loci was reduced 7- to 10-fold under anaerobic conditions. In addition, expression of the betB and betT loci was reduced when the cells were grown in high osmolarity at 16 degrees C. These studies demonstrate that the expression of the bet genes is under the control of several environmental stimuli.

Metabolism of [14C]-Monoethanolamine in Hordeum vulgare

The metabolism of [14C]-monothanolamine (EA) and its hydrochloride was investigated in long term experiments with intact plants of spring barley (Hordeum vulgare L.) during ontogenesis. The metabolism of EA applied as the free base (0.67 mg per plant) to an unwounded part of the fifth leaf of the main shoot proceeds relatively quickly. To a great extent, the EA taken up and translocated out of the feeding leaf was converted to volatile compounds during ontogenesis. The remaining amounts are incorporated into lipid soluble substances or metabolized via choline to glycine betaine. Wilting barley plants show a higher capacity for converting exogenous EA via the betaine pathway. However, there is no proportionality between [14C] -betaine synthesis from [14C]-EA and betaine accurrmulation. There are remarkable differences between EA and its hydrochloride with respect to metabolism. The hydrochloride is not converted to volatile compounds and glycine betaine is the main metabolite. The betaine pathway is only of minor importance for the metabolism of exogenous EA in turgid barley plants. The hydrochloride is also not completely metabolized in this way. Possible consequences are discussed.

An enzymatic synthesis of specifically radiolabelled derivatives of the common osmolyte, glycine betaine

A simple, rapid and efficient method has been developed for the synthesis of radiolabelled glycine betaine. Relatively inexpensive 14C- or 3H-labelled choline obtained from commercial sources is oxidized effectively using commercially available choline oxidase. Recoveries of >80% with >95% radiochemical purity are obtained using ion exchange chromatography. The addition of [ 14C-methyl]glycine betaine to samples from the water column of a hypersaline lake illustrate but one application for studies of methylated amine metabolism.

Infection with powdery mildew can enhance the accumulation of proline and glycinebetaine by salt stressed barley seedlings

The accumulation of proline and betaine by mildewed barley in response to salt stress (range 50 to 400 m M NaCl) and the effects of stress and infection on shoot dry weight were investigated using plants grown in sand culture. In view of the dependence of proline and betaine synthesis on nitrogen metabolism, tissue contents of nitrate, amino acids and ammonium ions were also determined. Uninfected and infected plants showed similar changes in water content and osmotic potential when similar concentrations of NaCl were applied. Concentrations of NaCl above 50 m M reduced shoot dry weight, and infection increased the severity of this reduction especially at the lower concentrations of NaCl. Above 50 m M NaCl, but not at 400 m M , salt-stressed, infected plants accumulated more proline in shoots than similarly salt-stressed controls. At 400 m M NaCl, shoots of uninfected and infected plants accumulated proline to the same extent. Infection had no effect at any stage on proline levels in roots, which remained low in salt-stressed plants, Infection only promoted the accumulation of betaine at 200 mm and 400 m M NaCl. Accumulations of nitrate were only detected in salt-stressed, infected plants at 400 m M NaCl. Infection promoted the salt-induced accumulation of ammonium ions by shoots, but had no effect on the amino acid content. It is suggested that increased proline and betaine accumulation may be advantageous to the plant if these osmotica are less readily taken up by the fungus than are simple alternative organic solutes.

Genetics of osmoregulation in Escherichia coli: Uptake and biosynthesis of organic osmolytes

Escherichia coli cells growing under osmotic stress activate systems for the transport or synthesis of several organic osmolytes. Glutamate and trehalose syntheses seem to represent mechanisms for achieving a low level of osmotic tolerance. The uptake and synthesis of betaines represent mechanisms of achieving a high level of osmotic tolerance. The osmotically-controlled ProP and ProU systems are involved in the uptake of glycine betaine and proline. However, glycine betaine synthesis occurs only in the presence of the precursor molecule choline. The osmotically controlled genes governing the high-affinity uptake of choline are located in close proximity to those encoding the dehydrogenases involved in the oxidation of choline to glycine betaine, but represent a different transcriptional unit. It is not known if each of these systems has its own osmotic sensor or whether a common osmotic sensor regulates all cell osmolytes.

Genetics of osmoregulation inEscherichia coli: Uptake and biosynthesis of organic osmolytes

Escherichia coli cells growing under osmotic stress activate systems for the transport or synthesis of several organic osmolytes. Glutamate and trehalose syntheses seem to represent mechanisms for achieving a low level of osmotic tolerance. The uptake and synthesis of betaines represent mechanisms of achieving a high level of osmotic tolerance. The osmotically-controlled ProP and ProU systems are involved in the uptake of glycine betaine and proline. However, glycine betaine synthesis occurs only in the presence of the precursor molecule choline. The osmotically controlled genes governing the high-affinity uptake of choline are located in close proximity to those encoding the dehydrogenases involved in the oxidation of choline to glycine betaine, but represent a different transcriptional unit. It is not known if each of these systems has its own osmotic sensor or whether a common osmotic sensor regulates all cell osmolytes.

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.

14C 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.

Water stress provokes a generalized increase in phosphatidylcholine turnover in barley leaves

Water and salt stress promote betaine accumulation in leaves of barley (Hordeum vulgare L.) by accelerating the de-novo synthesis of betaine, via choline. Previous radiotracer kinetic studies have implicated stress-enhanced turnover of the choline moiety of phosphatidylcholine (PC) as a major source of choline for betaine synthesis. Two approaches have therefore been followed to show whether stress-induced PC turnover is a cellor organelle-specific phenomenon, or a generalized one. In the first approach, [(3)H]ethanolamine of high specific activity was supplied to second leaves of unstressed and water-stressed barley plants; after 1 h, paired sections of tissue were excised from each leaf, one for extraction and analysis of [(3)H]metabolites and the other for autoradiography. The(3)H-activity remaining in the leaf tissue after washing out the water-soluble(3)H-metabolites during preparation for autoradiography was taken to be mainly in phospholipids. In unstressed leaves, [(3)H]phosphatidylethanolamine (PE) was the major labeled phospholipid, whereas there were approximately equal amounts of [(3)H]PE and [(3)H]PC in stressed leaves. At the light-microscope level, silver grains were associated with all living cells in both unstressed and stressed leaves; grains were concentrated in the cytoplasmic regions of highly vacuolate mesophyll cells, and were distributed throughout densely cytoplasmic vascular parenchyma. At the electron-microscope level, silver grains were not confined to any particular types of membranes in unstressed or stressed leaves. In the second approach, second leaves of stressed plants received a 1-h pulse of [(14)C]ethanolamine, and were then homogenized. The brei was subjected to sucrose density gradient centrifugation. The specific radioactivity of [(14)C]PC was quite similar in the gradient fractions, whether they contained microsomes or mitochondrial plus chloroplast membranes. We infer that stress does not enhance the turnover of any structurally discrete class of PC, but rather stimulates PC turnover in several or all classes of membranes in most cells of the leaf.

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.

Betaine Synthesis and Accumulation in Barley During Field Water‐Stress 1

The timing and extent of betaine accumulation by mature leaves of barley (Hordeum vulgare L.) were followed in irrigated (I) and non-irrigated (N-I) plots under rain-shelters. In the N-I crop, leaf water potential (/sup psi/leaf) began to fall at the five-leaf stage, continued to drop steadily until maturity, and reached a minimum of about -35 bars. Betaine accumulation started in the N-I crop about a week after the decline in /sup psi/leaf began and continued until about 10 days post-anthesis. The maximum betaine concentration attained by N-I leaves (100 ..mu..mol/g dry wt) was three times that in I leaves. Betaine accumulation by upper leaves was due mainly to de novo synthesis in these leaves, because: (1) there was little /sup 14/C-import into upper leaves when (/sup 14/C)betaine was applied to lower leaves, and (2) attached upper leaves of N-I plants rapidly converted supplied (/sup 14/C)ethanolamine to (/sup 14/C)betaine during the peak period of betaine accumulation. Phosphatidylcholine (PC) behaved as an intermediate in the conversion of (/sup 14/C)ethanolamine to betaine. The estimated peak metabolic cost of betaine biosynthesis via PC by stressed leaves (about 2 mg hexose/g dry wt/day) approached the cost of protein turnover in the same leaves (3 tomore » 5 mg hexose/g dry wt/day) as estimated from (/sup 3/H) lysine incorporation. In N-I plants, cessation of betaine synthesis preceded the onset of senescence by several days, indicating that continuous betaine production is not mandatory for leaf function at lowered /sup psi/leaf. These field results are consistent with an adaptive value for betaine accumulation in barley during prolonged water stress. A search for genetic variation in betaine-accumulating potential in barley is now warranted.« less

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.

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.