4-methyleneglutamine in peanut plants: dynamics of formation, levels, and turnover in relation to other free amino acids

Neither 4-methyleneglutamine nor 4-methyleneglutamic acid were found in free or bound form in ungerminated peanut seeds (Arachis hypogaea L.). Both, however, were formed soon after germination; whereas, 4-methyleneglutamic acid appeared slightly before 4-methyleneglutamine, the former remained at a low concentration while the level of 4-methyleneglutamine rose rapidly between 2 and 10 days of germination and declined slowly thereafter. Free proline and glutamine followed a pattern similar to 4-methyleneglutamine; on the other hand, asparagine increased for at least 20 days but other free amino acids remained at relatively low, constant levels. In mature peanut plants, 4-methyleneglutamine occurred in all parts except developing pods, was virtually the only free amino acid in xylem sap, and constituted about 70% of the total soluble nitrogen of sap. In contrast, 4-methyleneglutamic acid was found only in leaves and stems in highly variable amounts.     

4-Methyleneglutamic acid and 4-methyleneglutamine: isolation from extracts of peanut seedlings and determination by high-performance liquid chromatography

The non-protein amino acids, 4-methyleneglutamic acid and 4-methyleneglutamine, are isolated from aqueous extracts of peanut seedlings in good yield and high purity using a simple HCl-gradient elution from a column of cation-exchange resin followed, in some instances, by a gradient elution with acetic acid from a column of an anion-exchange resin. All of the 4-substituted glutamic acids commonly found in legume species are resolved by a combination of these two system. For analytical purposes, resolution of the acidic amino acids as their phenylthiocarbamoyl derivatives is achieved by HPLC but not by conventional ion-exchange amino acid analysis. Although 4-methyleneglutamine undergoes cyclic deamidation in acidic medium at a slower rate than glutamine, this reaction occurs to a significant extent at 22 degrees C but not a 4 degrees C during the cation-exchange chromatographic fractionation.     

Changes in γ-Methyleneglutamic acid, γ-Methyleneglutamine and other amino acids and amides during fruit growth of Tribulus terrestris

Abstract

Distribution and metabolism of γ-methyleneglutamic acid, γ-methyleneglutamine and other amino acids and amides has been studied during fruit growth of Tribulus terrestris. The largest concentration of free amino acids and amides has been observed in fruit stage 1. The marked decline in the amount of γ-methyleneglutamic acid and γ-methyleneglutamine after fruit stage 1 may indicate their rapid utilization along with asparagine and glutamine during fruit growth. In leaf and in different fruit growth stages, γ-methyleneglutamic acid dominated over γ-methyleneglutamine.     

Developmental and structural aspects of somatic embryos formed on medium containing 2,3,5-triiodobenzoic acid

Cotyledon explants of ginseng (Panax ginseng C.A. Meyer) zygotic embryos produced somatic embryos at a high rate (68%) on medium without any growth regulators. Under this culture condition, apparent polar somatic embryogenesis occurred near the basal-excised portion of the cotyledons. When the cotyledon explants were cultured on medium containing 2,3,5-triiodobenzoic acid (TIBA), an auxin polar-transport inhibitor, the frequency of somatic embryo formation markedly decreased and was completely inhibited on medium containing 20 μM TIBA. On medium containing 5–10 μM, somatic embryos developed sporadically on the surface of the cotyledons and had a normal embryo axis but jar-shaped cotyledons. Embryos with jar-shaped cotyledons were also observed to occur at a high frequency when the early globular embryos formed on hormone-free medium were transferred to medium containing 20 μM TIBA. From these results, it was deduced that endogenous auxin in the cotyledon explants plays an important role in the induction of somatic embryos and that the cotyledon development in somatic embryos is also related to the polar transport of endogenous auxin. Received: 11 October 1996 / Revised version received: 8 January 1997 / Accepted: 26 January 1997     

4-methyleneglutamine synthetase: a new amide synthetase present in germinating peanuts

Enzymatic activity which catalyzes the synthesis of 4-methyleneglutamine from 4-methyleneglutamic acid + ammonia was detected in and partially purified from cotyledons of peanut seeds germinated 5 to 7 days. This activity was separated from glutamine and asparagine synthetases by ammonium sulfate precipitation and DEAE-cellulose chromatography. The enzyme is distinct from these other amide synthetases in its substrate specificity, lack of amide/hydroxylamine exchange, and use of ammonium ion as amide donor together with formation of AMP from ATP. The activity is quite labile in solution, but is retained as a precipitate in ammonium sulfate or when frozen in 12.5% glycerol at -77 degrees C. This activity might be responsible for catalyzing the rapid synthesis of 4-methyleneglutamine which occurs in germinating peanuts.     

Repetitive somatic embryogenesis from peanut cultures in liquid medium

Summary A regeneration system based on repetitive somatic embryogenesis was developed for peanut (Arachis hypogaea L.). Embryogenic suspension cultures were initiated using individual somatic embryos induced from immature cotyledons cultured on a modified Murashige and Skoog medium containing 40 mg/l 2,4-D for 30 days. After transfer to a modified MS liquid medium, the somatic embryos produced masses of secondary and tertiary embryos which continued to proliferate following manual separation and subculture of the embryogenic clumps. The cultures exhibited exponential growth, and have been maintained for over one year without apparent loss of embryogenic potential. Further embryo development, germination, and conversion were achieved by placing embryo clumps onto hormone-free, solid medium. The inclusion of a desiccation period during embryo development enhanced conversion four-fold. Plants have been established in soil and appear to be phenotypically normal.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzylaminopurine - MSO Modified Murashige and Skoog basal medium - EM embryogenic masses     

Somatic embryogenesis from mature embryo-derived leaflets of peanut (Arachis Hypogaea L.)

Summary Embryogenic masses were obtained from immature leaves of peanut (Arachis hypogaea L.) cultured on a medium containing 20 mg/l 2,4-D. Somatic embryos developed from these masses following transfer to a medium containing 3 mg/l 2,4-D. The embryo morphology was quite variable. Following transfer to hormone-free medium, these embryos germinated. Shoot elongation was obtained in 25% of the embryos following transfer to a medium supplemented with 0.5 mg/l each of BAP and Kn. The plants grown in vitro by this method survived in sand:soil mixture and were grown to maturity.Abbreviations ABA abscisic acid - BAP 6-benzyl amino purine - 2,4-D 2,4 dichlorophenoxyacetic acid - GA₃ gibberellic acid - Kn kinetin - NAA 1-naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - Z zeatin     

Somatic embryogenesis from chickpea (Cicer arietinum L.) inmature cotyledons: The effect of zeatin, gibberellic acid and indole-3-butyric acid

Studies were conduced to test the effects of various cytokinins on somatic embryogenesis from chickpea (Cicer arietinum L.) immature cotyledons. Zeatin (13.7 μmol) added, to B5 basal medium, supplemented with 1.5 % sucrose and 0.2 μmol indole-3-acetic acid, was the most effective cytokinin. Lobular structures obtained from cotyledons cultures were transferred to B5 basal medium supplemented with gibberellic acid and indole-3-butyric acid at different concentrations. The most effective treatment was B5 medium containing 14.4 μmol gibberellic acid plus 1.0 μmol indole-3-butyric acid in which 42.8 % of lobular structures cultured formed normal somatic embryos. High conversion of embryos into plantlets (61.0–65.2 % embryos regenerated plants) was observed when germinated embryos were placed on plant development medium.     

4-methyleneglutamine amidohydrolase from peanut leaves : preparation, catalytic properties, and immunological responses of a highly purified form of the enzyme

4-Methyleneglutamine amidohydrolase has been extracted and purified over 1000-fold from 14-day-old peanut (Arachis hypogaea) leaves by modification of methods described previously. The purified enzyme shows two bands of activity and three to four bands of protein after electrophoresis on nondenaturing gels. Each of the active bands is readily eluted from gel slices and migrates to its original position on subsequent electrophoresis. Although they are electrophoretically distinct, the two forms of the enzyme are immunologically identical by Ouchterlony double-diffusion techniques and have similar catalytic properties. Activity toward glutamine that has a threefold lower V_max and a four-fold higher K_m value copurifies with MeGln aminohydrolase activity. 4-Methyleneglutamine and 4-methyleneglutamic acid inhibit the hydrolysis of glutamine while glutamine inhibits 4-methyleneglutamine hydrolysis, further indicating the identity of the activity toward both substrates. Amidohydrolase activity is stimulated up to threefold by preincubation with either ionic or non-ionic detergents (0.1%) and also by added proteins (0.5% bovine serum albumin or whole rabbit serum); it is inhibited 50% by 1 millimolar borate or the glutamine analog, albizziin (10 millimolar). Rabbit antiserum to the purified peanut enzyme cross-reacts with one or more proteins in extracts of some plants but not others; in no instance, however, was 4-methyleneglutamine amidohydrolase activity detected in other species. Overall, the results support the hypothesis that 4-methyleneglutamine supplies N, via its hydrolysis by the amidohydrolase, to the growing shoots of peanut plants, whereas glutamine hydrolysis is prevented by the prepon-derance of the preferred substrate. Some results also suggest that this amidohydrolase activity may be regulated by metabolites and/or by association with other cellular components.     

An unusual distribution of 4-substituted glutamic acids in Sophora japonica

High levels of 4-methyleneglutamine accumulate in the roots and leaves of Sophora japonica, but no detectable amounts of 4-methyleneglutamic acid and only trace quantities of 2-oxo-4-methyleneglutaric acid are seen. 4-Methylglutamic acid, however, is present in leaves and roots at a level 5–25% of that found for 4-methyleneglutamine; 2-oxo-4-methylglutaric acid is the most abundant keto acid detected in 28-day leaf extracts, but no 4-methylglutamine is seen. Transamination by pig heart glutamate: oxalacetate aminotransferase of the 2-oxo-4-methylglutaric acid that occurs in this species yields erythro-4-methylglutamic acid; the 2-oxo acid, therefore, has the (4R) configuration. The 4-methylglutamic acid isolated from this plant is also the erythro isomer and is probably of the (2S, 4R) configuration. This is the first report of the presence of 4-substituted glutamic acids in Sophora and the first instance where high levels of 4-methyleneglutamine are present in the absence of detectable levels of 4-methyleneglutamic acid.     

Purification and characterization of a novel 4-methyleneglutamine synthetase from germinated peanut cotyledons (Arachis hypogaea)

A newly detected amide synthetase, designated 4-methyleneglutamine synthetase, has been partially purified from extracts of 5- to 7-day germinated peanut cotyledons (Arachis hypogaea). Purification steps include fractionation with protamine sulfate and ammonium sulfate followed by column chromatography on Bio-Gel and DEAE-cellulose; synthetase purified over 300-fold is obtained. The enzyme has a molecular weight estimated to be approximately 250,000 and a broad pH optimum with maximal activity at approximately pH 7.5. Maximal rates of activity are obtained with NH+4 (Km = 3.7 mM) as the amide donor and the enzyme is highly specific for 4-methylene-L-glutamic acid (Km = 2.7 mM) as the amide acceptor. Product identification and stoichiometric studies establish the reaction catalyzed to be: 4-methyleneglutamic acid + NH4+ + ATP Mg2+----4-methyleneglutamine + AMP + PPi. PPi accumulates only when F- is added to inhibit pyrophosphatase activity present in synthetase preparations. This enzymatic activity is completely insensitive to the glutamine synthetase inhibitors, tabtoxinine-beta-lactam and F-, and is only partially inhibited by methionine sulfoximine. It is, however, inhibited by added pyrophosphate in the presence of F- as well as by certain divalent metal ions (other than Mg2+) including Hg2+, Ni2+, Mn2+, and Ca2+. All data obtained indicate that this newly detected synthetase is distinct from the well-known glutamine and asparagine synthetases.     

Optimization of somatic embryogenesis and embryo germination in soybean

Summary Somatic embryos of soybean [Glycine max (L.) Merr.] are induced on immature cotyledons explanted onto a medium containing moderately high levels of auxin. Germinability of embryos is related to morphologic normality, and both are reduced by excessive exposure to auxin during the induction process. Shoot meristem development was improved by reducing exposure of cotyledonary explants from 30 to 10 to 14 d on 10 mg/liter α-naphthaleneacetic acid (NAA). A 3-d exposure was sufficient to induce embryos, and embryo frequency was not significantly increased by exposures to NAA for more than 1 wk. Embryo frequency was enhanced, however, by transfer after 9 d to fresh medium containing 10 mg/liter NAA. Germination of morphologically normal embryos was achieved without growth regulators, after maturation for 1 mo. on hormone-free medium and desiccation for 1 wk in a sealed, dry container. This research was funded by Lubrizol Genetics, Inc., Madison, WI.     

Somatic embryogenesis and plant regeneration from leaflets of peanut,Arachis hypogaea

Somatic embryos were induced on peanut (Arachis hypogaea) leaflets from aseptically germinated embryo axes. Leaflet size influenced percent somatic embryogenesis; 5–8 mm long cut leaflets were superior to 2–3 mm long uncut leaflets. Maximum embryogenesis of 14.6% was obtained after a 15 d incubation on induction medium (modified MS with B5 vitamins, 30 g/l sucrose, 4 g/l Gel-Gro, 40 mg/l 2,4-D +0.2 mg/l kinetin) followed by transfer to a secondary medium with 5 mg/l 2,4-D+0.2 mg/l kinetin. Primary somatic embryos were fused along the axes with no distinct cotyledons, but secondary embryos had single axes with two cotyledons. Other treatments had lower percent embryogenesis, no secondary embryogenesis, and embryos with single axes with two cotyledons. Some somatic embryos converted into normal plants capable of greenhouse survival.Abbreviations MS Murashige and Skoog (1962) medium - B5 Gamborg et al. (1968) B5 medium - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6benzylaminopurine - NAA 1-naphthaleneacetic acid     

Direct somatic embryogenesis from axes of mature peanut embryos

Summary Plant regeneration via somatic embryogenesis was obtained in peanut (Arachis hypogaea L.) from axes of mature zygotic embryos. The area of greatest embryogenic activity was a 2-mm region adjacent to and encircling the epicotyl. Somatic embryogenesis was evaluated on Murashige and Skoog media supplemented with a variety of auxin treatments. Maximum production occurred on medium supplemented with 3 mg · liter⁻¹ 4-amino-3,5,6-trichloropicolinic acid. Explant cultures were transferred to half-strength medium supplemented with 1 mg · liter⁻¹ gibberellic acid for somatic embryo germination and early plantlet growth. Plantlets, transferred to soil, were placed in a greenhouse and grown to maturity.     

Glutamine Synthetase of Germinating Peanuts : PROPERTIES OF TWO CHROMATOGRAPHICALLY DISTINCT FORMS AND THEIR ACTIVITY TOWARD 4-METHYLENEGLUTAMIC ACID

Glutamine synthetase activity, extracted from an acetone powder of 7-day germinated peanuts (Arachis hypogaea L.), was precipitated by ammonium sulfate (40-60% saturation) and further purified by gel filtration and calcium phosphate gel treatment. When it was adsorbed to and subsequently eluted from a column of diethylaminoethyl-cellulose, two peaks of activity (designated glutamine synthetase 1 and 2) were obtained which were enriched 150- and 20-fold, respectively, over the initial extract. Glutamine synthetase 1 was present in ungerminated seeds and in the cotyledons during germination; glutamine synthetase 2 appeared during germination and was found largely in the developing plant. Compared with glutamine synthetase 2, glutamine synthetase 1 appeared to have a slightly smaller molecular weight and was more stable to heat and storage. The catalytic properties of the two forms were essentially the same. Whereas neither form catalyzed gamma-glutamyltransferase activity with 4-methyleneglutamine, both glutamine synthetases 1 and 2 catalyzed an ATP- and NH(4) (+)-dependent conversion of [(14)C]-4-methyleneglutamic acid to [(14)C]-4-methyleneglutamine, but the K(m) value for 4-methyleneglutamic acid was 10-fold greater and the V(max) only one-fourth that measured with l-glutamic acid. This is the first report of glutamine synthetase activity with 4-methyleneglutamic acid as substrate, although the level of this activity does not appear adequate to account for the rapid synthesis of 4-methyleneglutamine observed in germinating peanuts.     

Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.     

The Nitrogen Metabolism of Groundnut Plants: the Role of {gamma}-methyleneglutamine and {gamma}-methyleneglutamic Acid

The changes occurring in the nitrogenous compounds during thegrowth of groundnut seedlings in the dark and light were compared,particular attention being centred on the levels of -methyleneglutamine,the principal amide of these plants, and -methyleneglutamicacid. The distribution of amino acids and amides in the mainorgans of normal young and mature plants was also examined.Suggestions are made concerning the possible pathways of synthesisand the functions of -methyleneglutamic acid and -methyleneglutaminein groundnut plants.     

In vitro morphogenesis in zygotic embryo cultures of neem (<Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss.)

Immature zygotic embryo cultures of neem yielded highly regenerative cultures, with the response varying with the embryo stage at culture. Early dicotyledonous stage embryos were the most responsive followed by torpedo stage embryos. The embryo cultures differentiated three types of regenerants: somatic embryos (SEs), shoot buds and neomorphs. SEs exhibited morphological abnormalities such as pluricotyledony, fusion of cotyledons and absence of cotyledons. Although these SEs showed secondary embryogenesis, the occurrence of normal dicotyledonous embryos was extremely rare. On MS basal medium 3% of SEs developed a long tap root but a plumular shoot did not appear. However, it was possible to regenerate plantlets from immature zygotic embryo cultures of neem via neomorph formation and adventitious shoot bud formation. The transplantation survival of these plants was more than 80%.Abbreviations BAP 6-Benzylamino purine - CH Casein hydrolysate - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - SE Somatic embryoCommunicated by W. Harwood     

Soybean somatic embryogenesis: Effects of hormones and culture manipulations

Somatic embryos were induced in cultures of immature soybean (Glycine max (L.) Merr) embryos, or isolated cotyledons on MS modified medium supplemented with NAA and 2,4-D, BAP and ABA. When NAA and 2,4-D were compared at similar concentrations (25 and 23 M), 2,4-D produced larger number of somatic embryos, however, embryogenesis efficiency was improved in media containing NAA by using higher levels (100–150 M) of the auxin. Somatic embryo morphology varied with auxin type: NAA-induced embryos more closely resembled zygotic embryos than did 2,4-D-induced embryos. Additions of BAP or ABA to auxin-containing media had either no effect or reduced embryo production, although ABA altered the morphology of 2,4-D-induced embryos. In media containing both NAA and 2,4-D, the latter was dominant in terms of embryo morphology. The effects of subculture frequency and of transfers between 2,4-D and NAA media were investigated: Subculture frequency influenced mainly the frequency of normal embryos, while preculture on 2,4-D increased subsequent embryogenesis efficiency on NAA medium but reduced the frequency of normal embryos.Abbreviations Em^-2 s^-1 microEinsteins per square meter per second - NAA -naphthalene acetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid - ABA abscisic acid - BAP benzylamino purine This paper (No. 86-3-96), is published with the approval of the director of the Kentucky Agricultural Experiment Station.     

The effect of varying hormonal and precursor supplementations on levels of nicotine and related alkaloids in cell cultures ofNicotiana tabacum

Cell cultures ofNicotiana tabacum were grown on M & S medium containing 0–2.0 ppm of two auxins. Cellular nicotine was estimated for given auxin concentrations and highest levels determined. Feeding suspensions with amino acids either alone, or in combination with nicotinic acid and nicotinamide produced decresed nicotine levels. The presence of anatabine, anabasine, myosmine, anatalline and nicotelline in the cultures was established and it was noted that precursor feeding which decreased nicotine concentration caused elevated levels of the other alkaloids, notably anatabine.