Micropropagation of wild beet (Beta maritima) from inflorescence pieces

In order to investigate the regeneration of wild beet (Beta maritima) from inflorescence pieces, the effects of growth regulator, genotype, explant source and stage of plant development on adventitious shoot formation and rooting in vitro and subsequent transplanting in the glasshouse were tested. Inflorescence tips produced more adventitious shoots than sub-apical segments and the best micropropagation was achieved on a Murashige and Skoog (MS) medium supplemented with 1.0 mg l^–1 BAP. Addition of auxin was not beneficial. The induction rate of adventitious shoots was genotype-dependent and influenced by the stage of plant development. Adventitious shoots were produced from the base of the flower buds, i.e. from the receptacle, not from axils or stalks and only a few buds on inflorescence tip explants produced adventitious shoots. Rooting was increased by using a MS medium with 3% sucrose supplemented with 1.0 mg l^–1 NAA. There was no variation in leaf morphology of the transplants. This work shows that inflorescence tips can be used successfully as explants for in vitro multiplication of sugar beet and wild beet.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

Early effects of salt stress on the physiological and oxidative status of Cakile maritima (halophyte) and Arabidopsis thaliana (glycophyte)

Early changes in physiological and oxidative status induced by salt stress were monitored in two Brassicaceae plants differing in their tolerance to salinity, Cakile maritima (halophyte) and Arabidopsis thaliana (glycophyte). Growth response and antioxidant defense of C. maritima under 400 mM NaCl were compared with those of A. thaliana exposed to 100 mM NaCl. Salinity induced early growth reduction that is less pronounced in C. maritima than in A. thaliana. Maximum hydrogen peroxide (H₂O₂) level occurred in the leaves of both species 4 h after the onset of salt treatment. A rapid decline in H₂O₂ concentration was observed thereafter in C. maritima, whereas it remained high in A. thaliana. Correlatively, superoxide dismutase, catalase and peroxidase activities increased at 4 h of treatment in C. maritima and decreased thereafter. However, the activity of these enzymes remained higher in treated plants than that in controls, regardless of the duration of treatment, in A. thaliana. The concentrations of malondialdehyde (MDA) reached maximum values at 24 h of salt stress in both species. Again, MDA levels decreased later in C. maritima, but remained high in A. thaliana. The contents of α‐tocopherol remained constant during salt stress in C. maritima and decreased during the first 24 h of salt stress and then remained low in A. thaliana. The results clearly showed that C. maritima, in contrast to A. thaliana, can rapidly evolve physiological and antioxidant mechanisms to adapt to salt and manage the oxidative stress. This may explain, at least partially, the difference in salt tolerance between halophytes and glycophytes.     

Insect and wind pollination inUrginea maritima (Liliaceae)

Three co-existing pollination mechanisms are found inUrginea maritima: insect-, wind-, and self-pollination. The flowers exhibit a typical insect-pollination syndrome; they offer abundant exposed nectar as well as pollen. Out of the many different visitors only a few could be regarded as pollinators:Apis mellifera, Polistes gallicus, andVespa orientalis. Wind pollination also occurs and generally is responsible for self-pollination.It is argued that the development of extra wind-pollination accompanied by partial self-incompatibility is an adaptation to increase pollination in an unfavourable season (August–September), when insects are scarce.     

Purification and characterization of Thermotoga maritima homoserine transsuccinylase indicates it is a transacetylase

The methionine biosynthetic pathway found in bacteria is controlled at the first step, acylation of the γ-hydroxyl of homoserine. This reaction is catalyzed by one of two unique enzymes, homoserine transacetylase or homoserine transsuccinylase, which have no amino acid sequence similarity. We cloned, expressed, and purified homoserine transsuccinylase from the thermophilic bacterium Thermotoga maritima. Substrate specificity experiments demonstrated that acetyl-coenzyme A (CoA) is the preferred acyl donor and is used at least 30-fold more efficiently than succinyl-CoA. Steady-state kinetic experiments confirm that the enzyme utilizes a ping-pong kinetic mechanism in which the acetate group of acetyl-CoA is initially transferred to an enzyme nucleophile before subsequent transfer to homoserine. The maximal velocity, V/K _acetyl-CoA and V/K _homoserine, all exhibited bell-shaped pH curves with apparent pKs of 6.0–6.9 and 8.2–8.8. The enzyme was inactivated by iodoacetamide in a pH-dependent manner, with an apparent pK of 6.3, suggesting the presence of an active-site cysteine residue which forms an acetyl-enzyme thioester intermediate during catalytic turnover, similar to observations with other transsuccinylases. In addition, the enzyme is highly stable at elevated temperatures, maintaining full activity at 70°C. Taken together, these data suggest that the T. maritima enzyme functions biochemically as a transacetylase, despite having the sequence of a transsuccinylase.     

Morpho-anatomical features of the leaves of the mediterranean geophyteUrginea maritima (L) baker (Liliaceae)

The morpho-anatomy and histochemistry of the hysteranthous leaf ofUrginea maritima (L.) Baker and its adaptive strategies to the Mediterranean climate were investigated. The leaf ofU. maritima is 714 μm thick and possesses moderate specific leaf mass (8.564 mg cm^-2) and low tissue density (136.5 mg cm^-3). The epidermal cells are compactly arranged and covered with cuticle. The average density of stomata in lower epidermis is higher than that of the upper one. The mesophyll cells occupy 52.96% of the total volume of the leaf, while the mesophyll intercellular spaces and the air spaces occupy 30.41%. Idioblastic cells containing raphide bundles and different phenotypes of crystalloid inclusions, embedded in polysaccharides, occur in the lower side of the mesophyll. The presence of oil droplets and lipids is evident. Bundle sheath cells are hardly visible with no chloroplasts which are a pronounced C₃ plant character. Plastids containing protein crystalloid inclusions are abundant in the protophloem sieve elements.U. maritima, a deciduous plant, possesses leaves with mesophytic characters, in order to optimize its adaptation to the seasonal fluctuation of environmental conditions of the Mediterranean climate.     

Isolation and functional characterization of polyunsaturated fatty acid elongase (AsELOVL5) gene from black seabream (Acanthopagrus schlegelii)

To identify the genes encoding fatty acid elongases for the biosynthesis of polyunsaturated fatty acids (PUFAs), we isolated a cDNA via degenerate PCR and RACE-PCR from Acanthopagrus schlegelii with a high similarity to the ELOVL5-like elongases of mammals and fishes. This gene is termed AsELOVL5 and encodes a 294 amino acid protein. When AsELOVL5 was expressed in Saccharomyces cerevisiae, it conferred an ability to elongate γ-linolenic acid (18:3 n−6) to di-homo-γ-linolenic acid (20:3 n−6). In addition, the transformed cells converted arachidonic acid (20:4 n−6) and eicosapentaenpic acid (20:5 n−3) to docosatetraenoic acid (22:4 n−6) and docosapentaenoic acid (22:5 n−3), respectively. These results indicate that the AsELOVL5 gene encodes a long-chain fatty acid elongase capable of elongating C₁₈Δ6/C₂₀Δ5 but not C₂₂ PUFA substrates.     

Carrier design: Conformational studies of amino acid (X) and oligopeptide (X-DL-Alam) substituted poly(L-lysine)

The present study was undertaken to examine the influence of the reversal of the sidechain sequential order on the conformation of branched polypeptides. At the same time, the influence of the optically active amino acid joined directly to the poly (L -Lys) backbone and the DL -Ala oligomer grafted as chain-terminating fragment were separately analyzed. Therefore two sets of polypeptides were synthesized corresponding to the general formula poly [Lys-(X_i,)] (XK) and poly[Lys-(DL -Ala_m-X_i)] (AXK) when X = Ala, D -Ala, Leu, D -Leu, Phe, D -Phe, Ile, Pro, Glu.,D -Glu, or His. For coupling amino acid X to polylysine, three types of active ester methods were compared: the use of pentafluorophenyl or pentachlorophenyl ester, and the effect of the addition of an equimolar amount of 1-hydroxybenzotriazole. After cleavage of protecting groups, AXK polypeptides were synthesized by grafting short oligo (DL -Ala) chains to XK by using N-carboxy-DL -Ala anhydride. The CD measurements performed in water solutions of various pH values and ionic strengths were used for classification of the polypeptide conformations as either ordered (helical) or unordered. Different from what was observed with the unsubstituted poly (L -Lys), poly[Lys-(X_i)] type polypeptides can adopt ordered structure even under nearly physiological conditions (pH 7.3, 0.2M NaCl). These data suggest that the introduction of amino acid residue with either (ar) alkyl side chain (Ala, Leu, Phe) or negatively charged side chain (Glu) promotes markedly the formation of ordered structure. Comparison of chiroptical properties of poly [Lys- (DL -Ala_m-X_i)] and of poly [Lys- (X_i)] reveals that side-chain interactions play an important role in the stabilization of ordered solution conformation of AXK type branched polypeptides. The results give rather conclusive evidence that not only hydrophobic interactions, but also ionic attraction, can be involved in the formation and stabilization of helical conformation of branched polypeptides. © 1993 John Wiley & Sons, Inc.     

Salt-stimulated Phosphoenolpyruvate Carboxylase in Cakile maritima

The effects of NaCl and other salts, in vivo and in vitro, on the activity of phosphoenolpyruvate carboxylase from the coastal C₃ halophyte Cakile maritima Scop, were investigated. Plants grown with 100 mM NaCl in their growth medium yielded some 30% higher rates of phosphoenolpyruvate carboxylase activity than did salt-depleted plants. Activity of the enzyme was stimulated when NaCl was added to the reaction mixture in concentrations of up to 200 mM. The magnitude of this in vitro stimulation was similar for plants grown in the presence or absence of NaCl. The effect seems to be caused by chloride rather than by sodium ions.     

Effects of NaCl and Na2SO4 on Water Relations ofPlantago maritima (L.) andPlantago lanceolata (L.)

Transpiration and water absorption rates, stomatal and cuticular resistances to water vapour diffusion, were measured onPlantago maritima (halophyte) andPlantago lanceolata (glycophyte) grown in the presence of NaCl or Na₂SO₄. Water absorption was reduced in the presence of Na₂SO₄ and transpiration rate was increased when NaCl was added to the nutrient solution. The glycophyte daily water balance was more disturbed than that of the halophyte in the presence of Na₂SO₄.     

A novel bifunctional aspartate kinase-homoserine dehydrogenase from the hyperthermophilic bacterium,Thermotoga maritima

ABSTRACT

The orientation of the three domains in the bifunctional aspartate kinase-homoserine dehydrogenase (AK-HseDH) homologue found in Thermotoga maritima totally differs from those observed in previously known AK-HseDHs; the domains line up in the order HseDH, AK, and regulatory domain. In the present study, the enzyme produced in Escherichia coli was characterized. The enzyme exhibited substantial activities of both AK and HseDH. L-Threonine inhibits AK activity in a cooperative manner, similar to that of Arabidopsis thaliana AK-HseDH. However, the concentration required to inhibit the activity was much lower (K_0.5 = 37 μM) than that needed to inhibit the A. thaliana enzyme (K_0.5 = 500 μM). In contrast to A. thaliana AK-HseDH, Hse oxidation of the T. maritima enzyme was almost impervious to inhibition by L-threonine. Amino acid sequence comparison indicates that the distinctive sequence of the regulatory domain in T. maritima AK-HseDH is likely responsible for the unique sensitivity to L-threonine.

Abbreviations: AK: aspartate kinase; HseDH: homoserine dehydrogenase; AK–HseDH: bifunctional aspartate kinase–homoserine dehydrogenase; AsaDH: aspartate–β–semialdehyde dehydrogenase; ACT: aspartate kinases (A), chorismate mutases (C), and prephenate dehydrogenases (TyrA, T).     

Block oligopeptides (L-lysyl)m-(L-alanyl) L-tyrosyl-(L-alanyl)n- (L-lysyl)m. I. Synthesis through fragment condensation and characterization

Model peptides containing one aromatic residue were synthesized and characterized in order to investigate their interactions with polynucleotides. Chromatographically pure block oligopeptides (L -alysyl)_m-(L -alanyl)_n- L -tyrosyl- (L -alanyl)_n, with n = 3 and m=3 or 6, were prepared by fragments condensation using the mixed anhydride method. The protected fragments were prepared by stepwise addition of amino acid residues through the dicyclohexylcarbodiimide method. The purity of the intermediate coupling product was analyzed by gradient elution chromotography on carboxylmethylcellulose. Both block oligopeptides were isolated by preparative chromatography on carboxymethylcellulose. The different features of these syntheses are discussed.     

Synthesis of (R)-2-phenylpropanoic acid from its racemate through an isomerase-involving reaction by Nocardia diaphanozonaria

(R)-2-Phenylpropanoic acid was synthesized from the racemic acid through an isomerization reaction involving resting cells of Nocardia diaphanozonaria JCM3208. The isomerization activity of the cells was enhanced 25-fold by adding 5.5 mM racemic 2-phenylpropanoic acid to the culture medium. When 5 mM racemic 2-phenylpropanoic acid was included in the reaction mixture (4 ml) containing resting cells (100 mg dry cell wt) in 25 mM K₂HPO₄/KH₂PO₄ buffer (pH 7.0) at 30 °C for 8 h, 4.56 mM (R)-2-phenylpropanoic acid (95.8% e.e.) was formed with a 91% molar conversion yield.     

Plant defense approach of Bacillus subtilis (BERA 71) against Macrophomina phaseolina (Tassi) Goid in mung bean

This study was aimed to elucidate the mitigation mechanism of an endophytic bacterium, Bacillus subtilis (BERA 71) against Macrophomina phaseolina (Tassi) Goid disease in mung bean. M. phaseolina reduced the plant growth by inducing disease, hydrogen peroxide (H₂O₂) and lipid peroxidation. The inoculation of B. subtilis to diseased plants increased chlorophyll, ascorbic acids, and superoxide dismutase, catalase, peroxidase, ascorbate peroxidase and glutathione reductase activities, and while inhibited H₂O₂ and lipid peroxidation for enhancing plant growth. In addition, B. subtilis association in plants mitigated the M. phaseolina infection due to increase of indole acetic acids and indole butyric acid, and also a decrease of abscisic acid. However, the nutrients (N, K, Ca, Mg, Zn, Cu, Mn and Fe) were increased, except Na in M. phaseolina diseased plants treated with B. subtilis. The result of this study suggests that B. subtilis interaction with plants can modulate the metabolism of pigments, hormones, antioxidants and nutrients against M. phaseolina to induce disease resistance in mung bean.     

Synthesis and conformational study of sequential polypeptides, (L-ala-L-val-gly)n and (L-val-L-ala-gly)n.

As an approach for elucidating the role of sequences of amino acids in protein structures, model polypeptides having the same composition but different sequences of amino acids, (L -Ala-L -Val-Gly)_n and (L -Val-L -Ala-Gly)_n, have been prepared by the method involving facile monomer synthesis using N-carboxy α-amino acid anhydrides and N-hydroxysuccinimide esters. The yields and the molecular weights of the polypeptides formed by polycondensation do not depend on the monomer concentrations, but on the sequences of the amino acids in the monomers. Infrared spectra in the solid state showed that (L -Ala-L -Val-Gly)_n can take the α-helical conformation but (L -Val-L -Ala-Gly)_n cannot. The results suggest that the conformations of polypeptides are influenced by the sequences of the amino acids in the polypeptides.     

Purification,characterisation and amino acid composition of the putative moult-inhibiting hormone (MIH) ofCarcinus maenas (Crustacea,Decapoda)

Summary Using a Y-organ in vitro assay to measure repression of ecdysteroid synthesis in the presence of putative moult-inhibiting hormone (MIH), in conjunction with HPLC separation of sinus gland neuropeptides ofCarcinus maenas, it was found that both the hyperglycemic hormone (CHH) and a novel peptide (argued to represent the MIH) inhibited ecdysteroid synthesis. The latter was purified to homogeneity, and amino acid analysis showed that it is a 61 residue peptide (minimum molecular mass 7,200 Da) with the following amino acid composition: Asx₉; Thr₂; Ser₂; Glx₇; Pro₁; Gly₄; Ala₂; 1/2 Cys₄; Val₄; Met₁; Ile₃; Leu₅; Tyr₁; Phe₃; His₃; Trp₂; Lys₂; Arg₆. The N-terminus appears to be blocked. MIH is at least 20 times more potent than CHH in repressing ecdysteroid synthesis and is active at concentrations of less than 250 pmol/l. There may be structural similarities between CHH and MIH, howeve, MIH displays no CHH radioimmunoreactivity or hyperglycemic activity. The physiological significance of CHH in controlling ecdysteroid titres is not known.Abbreviations CHH hyperglycemic hormone - MIH moult inhibiting hormone - PAGE polyacrylamide gel electrophoresis - RIA radioimmunoassay - SDS sodium dodecyl sulfate - SG smus gland(s) - SGE sinus gland equivalent - TFA trifluoroacetic acid     

Parsing redox potentials of five ferredoxins found within Thermotoga maritima

Most organisms contain multiple soluble protein‐based redox carriers such as members of the ferredoxin (Fd) family, that contain one or more iron–sulfur clusters. The potential redundancy of Fd proteins is poorly understood, particularly in connection to the ability of Fd proteins to deliver reducing equivalents to members of the “radical SAM,” or S‐adenosylmethionine radical enzyme (ARE) superfamily, where the activity of all known AREs requires that an essential iron–sulfur cluster bound by the enzyme be reduced to the catalytically relevant [Fe₄S₄]¹⁺ oxidation state. As it is still unclear whether a single Fd in a given organism is specific to individual redox partners, we have examined the five Fd proteins found within Thermotoga maritima via direct electrochemistry, to compare them in a side‐by‐side fashion for the first time. While a single [Fe₄S₄]‐cluster bearing Fd (TM0927) has a potential of ?420 mV, the other four 2x[Fe₄S₄]‐bearing Fds (TM1175, TM1289, TM1533, and TM1815) have potentials that vary significantly, including cases where the two clusters of the same Fd are essentially coincident (e.g., TM1175) and those where the potentials are well separate (TM1815).     

Oxygen concentration affects nodule anatomy and nitrogenase activity of Alnus maritima

Alnus maritima is a shrub that associates with N₂-fixing Frankia in the wetlands in which it is native. Despite low concentrations in waterlogged soils, O₂ is critical to the maintenance of this symbiosis, and Frankia-infected nodules exist on roots of plants in native stands. The objective of the present study was to determine how root-zone O₂ concentration influences N₂ fixation and the anatomy of nodules on A. maritima. Root zones of plants inoculated with soil from native stands were exposed to eight O₂ concentrations. Nitrogenase activity increased with increasing O₂ concentration. Photosynthetic rate, plant dry mass, leaf N content, and nodule fresh mass were maximal in plants maintained with 15–25% O₂ in the root zone. Nodule counts were maximal on roots maintained at 10% and above 25% O₂, and nodules that developed at ≤ 2% O₂ were < 2 mm in diameter and single-lobed. Mean total area of air spaces within nodules decreased, and mean area per space increased, with increasing O₂ concentration. Seasonal and O₂-dependent nodule pigmentation was observed. Our data illustrate that O₂ is critical to the development of functional symbioses, and that nodules of this species, which are submersed in nature, possess mechanisms for responding to their low-O₂ environment.     

Production of isomeric 9,10,13 (9,12,13)-trihydroxy-11E (10E)-octadecenoic acid from linoleic acid by Pseudomonas aeruginosa PR3

Trihydroxy unsaturated fatty acids with 18 carbons have been reported as plant self-defense substances. Their production in nature is rare and is found mainly in plant systems. Previously, we reported that a new bacterial isolate, Pseudomonas aeruginosa PR3, converted oleic acid and ricinoleic acid to 7,10-dihydroxy-8(E)-octadecenoic acid and 7,10,12-trihydroxy-8(E)-octadecenoic acid, respectively. Here we report that strain PR3 converted linoleic acid to two compounds: 9,10,13-trihydroxy-11(E)-octadecenoic acid (9,10,13-THOD) and 9,12,13-trihydroxy-10(E)-octadecenoic acid (9,12,13-THOD). Stereochemical analyses showed the presence of 16 different diastereomers — the maximum number possible. The optimum reaction temperature and pH for THOD production were 30°C and 7.0, respectively. The optimum linoleic acid concentration was 10 mg/ml. The most effective single carbon and nitrogen sources were glucose and sodium glutamate, respectively. However, when a mixture of yeast extract (0.05%), (NH₄)₂HPO₄ (0.2%), and NH₄NO₃ (0.1%) was used as the nitrogen source, THOD production was higher by 8.3% than when sodium glutamate was the nitrogen source. Maximum production of total THOD with 44% conversion of substrate was achieved at 72 h of incubation, after which THOD production plateaued up to 240 h. THOD production and cell growth increased in parallel with glucose concentration up to 0.3%, after which cell growth reached its maximum and THOD production did not increase. These results suggested that THODs were not metabolized by strain PR3. This is the first report of microbial production of 9,10,13- and 9,12,13-THOD from linoleic acid. Journal of Industrial Microbiology & Biotechnology (2000) 25, 109–115. Received 18 March 2000/ Accepted in revised form 09 June 2000     

Conformational properties of the sequential polyproline analogs poly(Pro-Aze-Pro) and poly(Aze-Pro-Aze)

The lack of the positive band at around 226 nm in the CD spectra of poly(prolyl-azetidine-2-carbonyl-proline) in trifluoroethanol and of poly(azetidine-2-carbonyl-prolyl-azetidine-2-carboxylic) acid in F₃EtOH and water, the hyperchromism of the absorption maximum at about 202 nm, and the extremely small intensity of the C^β-Pro, C^γ-Pro, and C^β-Aze signals for the cis peptide bonds in the ¹³C nmr spectrum of poly(Pro-Aze-Pro) in F₃EtOH indicate that both polyproline analogs exist as disordered chains in this solvent, the trans peptide group being maintained. The disordering of the chains is attributed to an increase in the accessible range of ψ due to the reduced dimensions of the square ring of L -azetidine-2-carboxylic acid residue relative to the pyrrolidine ring of proline and to strong interactions of the haloalcohol with the peptide groups of the chains.     

Synergism between Metarhizium anisopliae (Deuteromycota: Hyphomycetes) and Boric Acid against the German Cockroach (Dictyoptera: Blattellidae)

Mortality of German cockroaches, Blattella germanica (L.), caused by Metarhizium anisopliae (Metschnikoff) Sorokin strain AC-1 alone and in combination with different formulations of boric acid, was evaluated in laboratory bioassays. Topical application of M. anisopliae alone (8.96 × 10⁹ conidia/m²) required 28 days to cause >92% cockroach mortality (LT₅₀ = 10 days). In contrast, in combination with boric acid (topically applied as a dust or in drinking water), M. anisopliae killed cockroaches significantly faster than without boric acid. M. anisopliae conidial dust (8.96 × 10⁸ conidia/m²) with either 12.5% (w/w) boric acid dust or 0.1% (w/v) boric acid in drinking water killed 100% of the cockroaches in only 8 days (LT₅₀ = 5 days) and 10 days (LT₅₀ = 6 days), respectively, without compromising the fungus emergence from cadavers. Replacement of M. anisopliae with flour dust or heat-killed M. anisopliae conidia eliminated this effect, demonstrating that it was not the consequence of greater boric acid ingestion due to more extensive cockroach grooming upon exposure to M. anisopliae conidia. Moreover, injections of a low dose of M. anisopliae, which caused only 30% mortality, together with sublethal concentrations of boric acid into the cockroach hemocoel resulted in a doubling of mortality. Statistical analysis demonstrated a synergistic interaction between these two insecticides.