Amino acids in seeds and seedlings of the genus Lens

The amino acid content of seeds and 4-day-old seedlings were studied in five species of lentil: Lens culinaris, L. orientalis, L. ervoides, L. nigricans and L. odemensis. Free amino acid and also total protein amino acid content after HCl hydrolysis were determined by HPLC. The nonprotein UV-absorbing amino acids were determined by capillary zone electrophoresis (CZE). The content of free protein amino acids in seeds varied among species and increased dramatically after germination. Asparagine is quantitatively most important in both seed and seedling. The content of free nonprotein amino acids is variable in seeds and seedlings. gamma-Hydroxyarginine, gamma-hydroxyornithine, alpha-aminobutyric acid and taurine were found in both seeds and seedlings. Homoarginine was found in four species but not in L. orientalis while gamma-aminobutyric acid (GABA), alpha-aminoadipic acid (alpha-aaa) and three isoxazolinone derivatives: beta-(isoxazolin-5-on-2-yl)-alanine (BIA), gamma-glutamyl-BIA (gamma-glu-BIA) and 2-carboxymethyl-isoxazolin-5-one (CMI) were found exclusively in the seedlings. CMI was identified for the first time in lentil species. Lathyrine, beta-(2-amino-pyrimidine-4-yl)-alanine, which was reported to be in the seeds of some Lathyrus species was confirmed to be present also in the seedling of L. culinaris (trace amount), L. nigricans and L. odemensis. Trigonelline (N-methyl-nicotinic acid), a plant hormone, is present both in seeds and seedlings in different concentrations except in L. ervoides. The different combination of nonprotein amino acids among the species gives indication of their genetic relationship and might partly explain the varying compatibility for interspecies crossing.     

Nonprotein amino acid composition of flatpea (Lathyrus sylvestris L.) as affected by ethephon seed treatments and seedling fertilization

Use of flatpea (Lathyrus sylvestris L.) as a forage is limited because of nonuniform seed germination and the potentially toxic effects of 2,4-diaminobutyric acid (A₂bu), a nonprotein amino acid found in seeds and vegetative tissues. The effects of ethephon (2-chloroethyl phosphonic acid) on seed germination, amino acid leachates of seeds, and amino acid composition (particularly A₂bu) of seedlings were investigated. Germination of flatpea seeds, imbibed for 16 h in 0, 100, 200, 400, 800, and 1600 mg/L ethephon, did not differ, but amino acid leachates tended to increase up to 200 mg/L ethephon and then decline at higher concentrations. The major amino acid constituents in leachates were A₂bu, 4-aminobutyric acid (Abu), and homoserine (Hse). Dry matter accumulation of seedlings grown from ethephon-treated seeds was reduced for second cuttings grown from ethephon-treated seeds and high nitrogen grown plants. During regrowth, free amino acid accumulation was most pronounced in leaves of plants supplied with high nitrogen. The most abundant free amino acids in flatpea tissues were the same as those in seed leachates, but concentration and relative abundance varied with nitrogen level, plant part, and ethephon treatment. Results suggest that ethephon seed treatments can have persistent effects on the growth and amino acid composition of flatpea seedlings grown under different nitrogen regimes.     

Nonprotein amino acid composition of flatpea (Lathyrus sylvestris L.) as affected by ethephon seed treatments and seedling fertilization

Use of flatpea (Lathyrus sylvestris L.) as a forage is limited because of nonuniform seed germination and the potentially toxic effects of 2,4-diaminobutyric acid (A₂bu), a nonprotein amino acid found in seeds and vegetative tissues. The effects of ethephon (2-chloroethyl phosphonic acid) on seed germination, amino acid leachates of seeds, and amino acid composition (particularly A₂bu) of seedlings were investigated. Germination of flatpea seeds, imbibed for 16 h in 0, 100, 200, 400, 800, and 1600 mg/L ethephon, did not differ, but amino acid leachates tended to increase up to 200 mg/L ethephon and then decline at higher concentrations. The major amino acid constituents in leachates were A₂bu, 4-aminobutyric acid (Abu), and homoserine (Hse). Dry matter accumulation of seedlings grown from ethephon-treated seeds was reduced for second cuttings grown from ethephon-treated seeds and high nitrogen grown plants. During regrowth, free amino acid accumulation was most pronounced in leaves of plants supplied with high nitrogen. The most abundant free amino acids in flatpea tissues were the same as those in seed leachates, but concentration and relative abundance varied with nitrogen level, plant part, and ethephon treatment. Results suggest that ethephon seed treatments can have persistent effects on the growth and amino acid composition of flatpea seedlings grown under different nitrogen regimes.     

Developmental changes of free amino acids in bovine fetal fluids with gestational age and the interrelationships between the amino acid concentrations in the fluid compartments.

Thirty ninhydrin-positive compounds were determined in the sera, amniotic fluid, stomach content and allantoic fluid from 29 bovine fetuses of various gestational ages. Fetal serum was found to contain about 3-fold higher concentrations of free amino acids (FAA) than maternal serum, and allantoic fluid contained about 3-fold higher concentration of FAA than fetal serum. Decreasing concentrations of FAA were found in serum as a function of the crown-rump length for the amino acids taurine, aspartic acid, threonine, serine, ornithine and lysine. Decreasing concentrations of FAA in allantoic fluid were found for threonine, alanine and ornithine, whereas increasing concentrations were found for phosphoserine and methionine as a function of the crown-rump length. Correlations were found between the concentrations of most amino acids in amniotic fluid and stomach content, but fewer correlations were found between the other fluid compartments. The transport of amino acids between compartments is discussed.     

Amino acids of Caesalpinia tinctoria and some allied species

Two previously unreported amino acids have been found to form major constituents of seed of Caesalpinia tinctoria; they have been characterized as 4-hydroxy-3-hydroxymethylphenylalanine and 3-hydroxymethylphenylalanine. The seed also contains a large amount of the imino acid baikiain. The amino acid content of seeds of this species is compared with that for 12 other species of Caesalpinia and 14 species in closely related genera.     

Accumulation of amino acids in muscle of perfused rat heart. Effect of insulin

1. Rat heart perfused with Krebs-Henseleit bicarbonate buffer released material containing ninhydrin-positive nitrogen, but the amount was less than that reported to be released by diaphragm; glucose, but not insulin, decreased the release of ninhydrin-positive nitrogen and increased the concentration of the same material in the intracellular water of heart. 2. When heart was perfused with a mixture of amino acids and glucose, there was actually a net uptake, and an increase in intracellular concentration, of ninhydrin-positive nitrogen. Changes in the concentration of ninhydrin-positive nitrogen did not accurately reflect changes in concentration of amino acids. 3. The effect of insulin on the actual concentration of individual amino acids in heart muscle was examined by perfusing the heart with a mixture of amino acids and other ninhydrin-positive substances in the same concentration as they are found in plasma. 4. The effect of insulin on the concentrations of amino acids in the medium and in the intracellular water of the heart was determined after perfusion for different periods of time. No clear or meaningful effect of insulin was observed, despite the fact that insulin significantly increased the accumulation, in each of the same hearts, of radioactivity from amino[(14)C]isobutyric acid.     

Partial purification and properties of a transamidinase from Lathyrus sativus seedlings. Involvement in homoarginine metabolism and amine interconversions.

A transamidinase was purified 463-fold from Lathyrus sativus seedlings by affinity chromatography on homoarginine--Sepharose. The enzyme exhibited a wide substrate specificity, and catalysed the reversible transfer of the amidino groups from donors such as arginine, homoarginine and canavanine to acceptors such as lysine, putrescine, agmatine, cadaverine and hydroxylamine. The enzyme could not be detected in the seeds, and attained the highest specific activity in the embryo axis on day 10 after seed germination. Its thiol nature was established by strong inhibition by several thiol blockers and thiol compounds in the presence of ferricyanide. In the absence of an exogenous acceptor, it exhibited weak hydrolytic activity towards arginine. It had apparent mol.wt. 210000, and exhibited Michaelis--Menten kinetics with Km 3.0 mM for arginine. Ornithine competitively inhibited the enzyme, with Ki 1.0 mM in the arginine--hydroxylamine amidino-transfer reaction. Conversion experiments with labelled compounds suggest that the enzyme is involved in homoarginine catabolism during the development of plant embryo to give rise to important amino acids and amine metabolites. Presumptive evidence is also provided for its involvement in the biosynthesis of the guanidino amino acid during seed development. The natural occurrence of arcain in L. sativus and mediation of its synthesis in vitro from agmatine by the transamidinase are demonstrated.     

Characteristics of Sugar, Amino Acid and Phosphate Release from the Seed Coat of Developing Seeds of Vicia faba and Pisum sativum

After removal of the embryo from developing seeds of Vicia fabaL. and Pisum sativum L., the ‘empty’ ovules werefilled with a standard solution (pH 5.5). Seed coat exudatesof both species were collected during relatively long experiments(up to about 12 h) and the concentration of sugar (mainly sucrose),amino acids and phosphate in the exudate measured. A discussionis presented on the amino acid/sugar ratio and the phosphate/sugarratio in the seed coat exudate. A pretreatment (15 min) withp-chloromercuribenzenesulphonic acid (PCMBS) reduced the releaseof sugar, amino acids and phosphate from broad bean seed coats.After excision of ‘empty’ ovules of Vicia faba andPisum sativum from the maternal plant, 2–4 h after thistreatment a strong difference became visible between sucroserelease from excised seed coats and sucrose release from attachedseed coats. Similarly, when the rate of phloem transport ofsucrose into an ‘empty’ ovule of Vicia faba or Pisumsativum was reduced by a sub-optimal mannitol concentrationin the solution, a reduced rate of sugar release from the seedcoat could be observed. Excision and treatment with a sub-optimalmannitol concentration reduced the release of amino acids toa lesser extent than for sucrose. These treatments did not reducethe rate of phosphate release from the seed coat. Key words: Seed development, Seed coat exudate, Phloem transport     

Effects of Nutrient Supply on Seed Growth of Field Bean (Vicia faba L.). 1. Effects of Different Sulphur and Methionine Regimes on Cotyledon Growth, Protein and Uncombined Amino Acids In vivo

The effects of exogenous methionine on growth of developingVicia faba seeds in vivo was studied. Methionine (51 or 102µmol in total) injected into the base of the pod overa period of 15 d (26–40 d after pollination) decreasedgrowth and protein accumulation in proximal seeds but increasedin distal seeds. Distal seeds of methionine injected pods accumulatedmore d. wt and protein than distal seeds of water-injected pods.However, on a pod basis methionine reduced overall seed growthand protein accumulation. Exogenous methionine caused a relative increase in legumin buta decrease in vicilin. Injected methionine also affected thecomposition of uncombined amino acids, especially those derivedfrom aspartic acid. In contrast, the amino acid compositionof the protein fraction did not change appreciably. The data suggest that seed growth is not limited by methioninebiosynthesis but seed protein composition is regulated by methionine. Vicia faba L., field bean, cotyledon, growth, in vitro culture, uncombined amino acids, protein composition, legumin, vicilin, methionine, sulphur     

Isoxazolin-5-one derivatives in Lathyrus odoratus during development and growth

Variations in the concentrations of isoxazolin-5-ones and of some non-protein amino acids in the dry seeds, seedlings and various parts of mature Lathyrus odoratus plants were examined. The lathyrogenic compounds α-amino-γ-(isoxazolin-5-on-2-yl)-butyric acid, 2-cyanoethyl-isoxazolin -5-one and γ-glutamyl-β-aminopropionitrile were major products during development and growth.     

银杏种子生长特性及其生理变化的研究

从纵径、横径、体积、重量等方面分析了银杏种子的生长特性及其生长过程中水分、糖类物质、脂肪酸、氨基酸的生理变化.结果表明,银杏种子的生长过程曲线为典型的单“S”型,种子纵径、横径、体积、重量、绝对含水量随生长过程呈“S”形变化.各种生理物质含量随种子生长过程呈规律性变化,且各有其特点.其中在种子生长后期糖类物质总量表现出上升的趋势,表明它们是种子中主要的营养储存物质.成熟种子含淀粉8.4%、葡萄糖6.7%、果糖4.2%、多糖0.02%、二糖0.01%;含肉豆蔻酸10.6%、棕榈烯酸4.1%、亚麻酸2.4%、硬脂酸1.9%、油酸1.1%、亚油酸0.4%.银杏种子富含15种氨基酸,成熟种子中总含量为1.56g·100g^-1FW,其中以赖氨酸、天门冬氨酸、丙氨酸、精氨酸、组氨酸、谷氨酸、异亮氨酸的含量较高,它们的含量依次为0.287%、0.163%、0.136%、0.133%、0.123%、0.115%、0.095%.     

The effect of some non-protein amino acids on pollen germination and pollen-tube growth in five species of the Vicieae

Summary The effects of canavanine, ,-diaminobutyric acid, homoarginine and lathyrine on the germination of pollen and on in-vitro growth of pollen tubes were studied in the following species: Lathyrus niger, L. silvestris, Vicia unijuga, Pisum sativum and Cicer arietinum.The effects of these non-protein amino acids depended on their quantity and on the plant species. Every amino acid had a promoting effect on germination and growth at some concentration in some species. Inhibition or promotion of pollen germination and pollen-tube growth were usually parallel. The stronger influence of some amino acid on growth than on germination may be due to slow penetration of the acid into the cell.Homoarginine and lathyrine had a promoting effect at all concentrations in L. niger, a species in which these amino acids occur naturally. In most other species they had, if anything, a very slight inhibitory effect, ,-Diaminobutyric acid and canavanine had the strongest inhibitory effects on the species studied. It seems possible that these amino acids are antimetabolites of common amino acids.It is obvious that non-protein amino acids can form effective hybridization barries, although the conditions in nature are more complex than in vitro. The ability to synthesize a new amino acid may therefore be of evolutionary significance in the isolation of new species and genera.     

Protein and free amino acids in field-grown cowpea seeds as affected by water stress at various growth stages

Summary This study was undertaken to evaluate water stress effects during vegetative, flowering, and podfilling stages of cowpea plants (Vigna unguiculata L.) grown under natural field conditions in southern California on seed yield and protein and free amino acid content of the cowpea seeds. The lowest concentration of N was found in the seeds of the control treatment plants while the seed yield from these treatments was the highest as compared with the N concentration and yield of seeds from plants subjected to water stress during flowering and podfilling stages. The concentration of N in the seeds was inversely related to the seed dry weight yield. Protein arginine,-threonine,-serine,-cystine,-valine,-methionine, and-isoleucine were significantly affected by water stress at the three growth stages. There was no consistent pattern in the effect of water stress on the individual amino acids. The sum of protein amino acids in the cowpea seeds was not significantly influenced by the various treatments since some of the protein amino acids increased and others decreased producing an averaging effect on the figures comprising the sums of the amino acids. Water stress during the flowering and pod-filling stages increased the free amino acid pool, and at the same time, inhibited incorporation of the amino acids into the protein chain-thus lowering the protein amino acid fraction simultaneously. With the exception of methionine plus cystine, the essential amino acids in the seeds were present at concentrations equal to or greater than recommended by the World Health Organization and FAO. It is of particular importance to note that the concentration of lysine in the cowpeas was substantially higher than that found in wheat grain. It is also important to note that the amount of essential amino acids per gram of protein was not measurably affected by the water stress treatments during any of the growth stages.     

Compartmentation of transport and transfer events in developing seeds

Developing seeds are net importers of organic and inorganic nutrients. Nutrients enter seeds through the maternal vascular system at relatively high concentrations in the phloem. They exit importing sieve elements via interconnecting plasmodesmata and, during subsequent symplasmic passage, are sequestered into labile storage pools (vacuoles; starch). Transporters function to retrieve nutrients leaked to the seed apoplasm during symplasmic passage. Maternal cells responsible for nutrient release to the seed apoplasm are characteristically located at the maternal/filial interface. Their plasma membranes are enriched in transport proteins and, in some species, these cells are modified to a transfer cell morphology. Apoplasmic volumes of seeds are relatively small, but contain high concentrations of sugars, potassium and a range of amino acids. Sucrose and amino acids are taken up from the seed apoplasm by one to two cell layers of filial tissues that juxtapose the maternal tissues. The plasma membranes of the uptake cells are enriched in sucrose and amino acid/H(+) transporters which co-localize with H(+)-ATPASES: In some species, these cells are modified to a transfer cell morphology. High densities of plasmodesmata support symplasmic delivery of accumulated nutrients to underlying storage cells where polymer formation (starch, protein) takes place. Hexoses, resulting from sucrose hydrolysis and leakage to the seed apoplasm, are retrieved by hexose/H(+) symporters.     

A comparative study of convicilin storage protein gene sequences in species of the tribe Vicieae.

Convicilins, a set of seed storage proteins, differ from vicilins, a related group of seed storage proteins, mainly because of the presence of the N-terminal extension, an additional sequence of amino acids in the sequence corresponding to the first exon. Convicilins have been described only in species of the legume tribe Vicieae. One or two genes for convicilins have been identified in most species of this tribe. The genus Pisum is the main exception, since two genes have been identified in most of its species. Thirty-four new convicilin gene sequences from 29 different species (Lathyrus, Lens, Pisum, and Vicia spp.) have been analyzed here. Convicilin gene sequences are generally organized in 6 exons, but in some instances one of the internal introns (2nd or 4th) is lost. In these 29 species, the N-terminal extension is formed by a stretch of 99 to 196 amino acids particularly rich in polar and charged amino acids (on average, it contains 29.43% glutamic acid and 15.38% arginine residues). This N-terminal extension has the characteristics of an intrinsically unstructured region (IUR), one of the categories of protein "degenerate sequences". A comparative analysis indicates that the N-terminal extension sequence has evolved faster than the surrounding sequence, which is common to all vicilins, and it evolved mainly through a series of duplications of short internal sequences and triplet expansions, the predominant one being GAA. This agrees with the evolution of IURs, which is faster than the evolution of surrounding sequences and is mainly due to replication slippage and unequal crossover recombination. Alternative maximum-likelihood trees of phylogenetic relationships among the 29 Vicieae species based on the convicilin exon sequences are presented and discussed.     

Ectopic expression of an amino acid transporter (VfAAP1) in seeds of Vicia narbonensis and pea increases storage proteins

Storage protein synthesis is dependent on available nitrogen in the seed, which may be controlled by amino acid import via specific transporters. To analyze their rate-limiting role for seed protein synthesis, a Vicia faba amino acid permease, VfAAP1, has been ectopically expressed in pea (Pisum sativum) and Vicia narbonensis seeds under the control of the legumin B4 promoter. In mature seeds, starch is unchanged but total nitrogen is 10% to 25% higher, which affects mainly globulin, vicilin, and legumin, rather than albumin synthesis. Transgenic seeds in vitro take up more [14C]-glutamine, indicating increased sink strength for amino acids. In addition, more [14C] is partitioned into proteins. Levels of total free amino acids in growing seeds are unchanged but with a shift toward higher relative abundance of asparagine, aspartate, glutamine, and glutamate. Hexoses are decreased, whereas metabolites of glycolysis and the tricarboxylic acid cycle are unchanged or slightly lower. Phosphoenolpyruvate carboxylase activity and the phosphoenolpyruvate carboxylase-to-pyruvate kinase ratios are higher in seeds of one and three lines, indicating increased anaplerotic fluxes. Increases of individual seed size by 20% to 30% and of vegetative biomass indicate growth responses probably due to improved nitrogen status. However, seed yield per plant was not altered. Root application of [15N] ammonia results in significantly higher label in transgenic seeds, as well as in stems and pods, and indicates stimulation of nitrogen root uptake. In summary, VfAAP1 expression increases seed sink strength for nitrogen, improves plant nitrogen status, and leads to higher seed protein. We conclude that seed protein synthesis is nitrogen limited and that seed uptake activity for nitrogen is rate limiting for storage protein synthesis.     

Single-column amino acid analysis of connective tissue proteins and related materials.

A single-column procedure is described which, in general, is useful for the amino acid analysis of any simple or complex protein, but in particular the procedure is useful for the separation and quantitation of the amino acids and amino sugars, if present, in collagen, elastin and related materials from a variety of connective tissues. In addition to the amino acids commonly found in proteins, the system resolves hydroxyproline, hydroxylysine, desmosine, isodesmosine, glucosamine, galactosamine and also a number of other ninhydrin-positive compounds ordinarily not found in acid hydrolysates of proteins. Chromatograms of the analysis of a synthetic mixture of amino acid standards and also collagen and elastin hydrolysates indicate the multiple use of the procedure and the nearbaseline separation of all the amino acids. The basic amino acids are well spread, thus providing flexibility for the isolation and identification of additional ninhydrin-positive components. The analysis, which requires approximately 2 hr and four sodium citrate buffers was performed with a Durrum (D-500) amino acid analyzer.     

Determination of 4-chloroindoleacetic acid methyl ester in Vicieae species by gas chromatography-mass spectrometry

The natural chlorinated auxin, 4-chloroindoleacetic acid methyl ester, was identified in immature seeds of Lathyrus sativus L., Lathyrus maritimus (L.) Bigel and Lathyrus odoratus L. by thin layer chromatography and gas chromatography-mass spectrometry. In immature seeds of Vicia sativa L. and Lens culinaris Medik. the hormone was identified by selected ion monitoring. The hormone was determined quantitatively using pentadeuterated 4-chloroindoleacetic acid methyl ester as internal standard. Contents varied from 1 mg/kg fresh weight in Lathyrus sativus to 0.02 mg/kg in Lens culinaris. Lathyrus maritimus also contained indoleacetic acid methyl ester (0.3 mg/kg) besides the chlorinated analogue.     

The effect of fluctuating salinity on the concentrations of free amino acids and ninhydrin-positive substances in the adductor muscles of eight species of bivalve molluscs

Eight species of bivalve molluscs were exposed both to gradual and abrupt salinity fluctuations and the changes in free amino acids and ninhydrin-positive substances in their adductor muscles measured. In all the species there was an initial rise in the concentration of ninhydrin positive substances when exposed to decreasing salinities. After acclimation for one week (14 cycles) to a 30 % sea-water minimum sinusoidal salinity regime there was no difference in the concentration of ninhydrin positive substances at high and low salinities in the adductor muscles of Mytilus edulis L. Together, the changes in taurine and non-essential amino acids alanine, aspartic acid, glutamic acid, and glycine largely accounted for the changes in the free amino-acid pool. It was found that ‘shell-closing’ mechanisms may result in changes in the free amino-acid pool brought about by reductive amination of Krebs cycle and other keto-acids under anaerobic conditions. It is suggested that ninhydrin-positive substances and free amino acids are used as osmotic effectors in marine bivalves exposed to constantly lowered salinities, but are not used for the same purpose in animals exposed to cyclic salinity changes.     

Chemical differences between seeds and elaiosomes indicate an adaptation to nutritional needs of ants

Ant-dispersed plants usually produce seeds with appendages (elaiosomes) as reward for ants. Plants that produce high-quality elaiosomes benefit because ants preferentially disperse their diaspores. We therefore hypothesized that seeds and elaiosomes differ in chemical composition in ways that make elaiosomes of high nutritional quality for ants, capable of providing essential dietary components that explain the increased fitness and higher gyne production documented for colonies with elaiosome consumption. To test the hypothesis we analysed the content and composition of lipids, amino acids, soluble carbohydrates, proteins and starch in seeds and elaiosomes of 15 central European ant-dispersed plants. After separating the different fractions, total lipids were determined gravimetrically, fatty acids and soluble carbohydrates were detected by gas chromatography (GC) and GC–mass spectrometry, free amino acids by an amino acid analyser while starch and protein were analysed photometrically. Seeds accumulated high molecular weight compounds such as proteins and starch, whereas elaiosomes accumulated more easily digestible low molecular weight compounds such as amino acids and monosaccharides. Analysis of similarities and similarity percentages analysis demonstrated that the composition of fatty acids, free amino acids and carbohydrates differed markedly between elaiosomes and seeds. The most important difference was in total amino acid content, which was on average 7.5 times higher in elaiosomes than in seeds. The difference was especially marked for the nitrogen-rich amino acid histidine. The availability of essential nutrients and, in some species, the higher nitrogen content in elaiosomes suggest that their nutritional value for larvae plays a key role in this interaction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.