Possible metabolic basis for the developmental anomaly observed in in vitro culture,called ‘vitreous plants’

Previous studies have shown that the development anomaly encountered in meristem culture, known as vitreous plants, is due to deficient lignin synthesis. This anomaly can be cured by addition of phloridzin to the culture medium. This study examines the activities of some enzymes involved in the synthesis of lignins and of flavonoids in normal and in vitreous plants of two apple cultivars. The results showed that all enzymes were consistently less active in the vitreous plants. This agrees with previous studies made on the hydroxycinnamate: CoA ligase activity in Prunus avium (L.) meristem-derived plants. The study on the substrate specificity of the enzyme demonstrates that while its activity is lower in the vitreous plant, its conformation is identical with that of the normal plant; the substrate that is specific to enzyme extracts of both sources is para-coumaric acid.     

Effect of various culture conditions on shoot multiplication and GC–MS analysis of <Emphasis Type="Italic">Plumbago zeylanica</Emphasis> accessions for plumbagin production

Plumbago zeylanica, a pharmaceutically important medicinal plant, contains a wide range of phytocompounds. Culture parameters like carbon source, nitrogen source, and culture media are essential for the development and growth of explants. In this investigation, the influence of various carbon sources (sucrose, glucose, and fructose at 3% concentration), nitrogen source (ammonium nitrate, sodium nitrate, and potassium nitrate) and plant tissue culture media (MS medium, Gamborg’s B5 medium, White medium and Nitsch medium) on shoot multiplication of five different accessions was studied. Optimum growth of all five accessions was observed in MS media containing 3% sucrose and ammonium nitrate as a source of carbon and nitrogen. Out of five accessions, IC-524441 showed the highest shoot multiplication. Further, methanolic extracts of all accessions (grown in MS media containing 3% sucrose and ammonium nitrate as nitrogen source) were prepared and comparison of extracts in DPPH assay indicated that accession number IC-524441 was the most effective free radical scavenging agent. Total phenolic, flavonoid and tannin content ranges were from 20 to 70 µg/ml, 40 to 100 µg/ml and 55 to 120 µg/ml, respectively, and the highest amount was found in accession number IC-524441. Sucrose and ammonium nitrate content may be responsible for increased antioxidant activity, flavonoids content, phenolic content, and tannin content in accession number IC-524441. GC–MS of ethyl acetate extract of all five accessions of P. zeylanica was conducted (grown in MS media containing 3% sucrose and ammonium nitrate as nitrogen source). GC–MS analysis of the aerial part showed the presence of various phytocompounds, which include 1,4-naphthalenedione, 3-eicosene, 5-eicosene, phthalic acid, o-anisic acid, thioctic acid, 1-octadecene, 5-t-butyl-cycloheptene, 2-benzoyl-1,2-dihydro-1-isoquinolinecarbonitrile, octadecanal, silane, 3-methoxy-2-methyl-2-(1-phenyl-ethylamino)-propionic acid, and 1-nonadecene. Accession number IC-524441 contains the highest amount of plumbagin, i.e. 14.19?±?0.5 µg/ml as compared to the others.     

Influence of ammonium and extracellular pH on the amino and organic acid contents of suspension culture cells of Acer pseudoplatanus

The effects of supplied ammonium and nitrate on the amino and organic acid contents and enzyme activities of cell suspension cultures of Acer pseudoplatanus L. were examined. Regardless of nitrogen source the pH of the culture medium strongly affected the malate and citrate contents of the cells; these organic acid pools declined at pH 5, but increased at pH 7 and 8. Over a period of two days, ammonium had little effect on the responses of the organic acid pool sizes to the pH of the medium. In contrast, ammonium had a strong influence on amino acid pool sizes, and this effect was dependent on the pH of the medium. At pH 5 there was no increase in cell ammonium or amino acid contents, but at higher pH values cellular ammonium content rose, accompanied by accumulation of glutamine, glutamate and asparagine. Over several days, supplied ammonium led to an increase in activity of glutamate dehydrogenase irrespective of any changes in internal ammonium and amino acid contents. If the pH of the medium was allowed to fall below pH 4 in the presence of ammonium, phosphoenolpyruvate (PEP) carboxylase activity declined to a very low value over several days; at higher pH, the activity of this enzyme, and that of NAD malic enzyme and NAD malate dehydrogenase, remained substantial irrespective of whether the nitrogen source was NH⁺₄ or NO-₃.     

Controlling vitrification of petunia in vitro

Summary Nodal segments from in vitro culturedPetunia hybrida were grown under varying cultural conditions. The origin of nodal explants influenced vitrification. Basal segments formed a higher percentage of vitreous shoots than did the upper nodes. A method was developed for including polyethylene glycol with Gelrite to obtain gelled media of varying water potentials. Media water potential from −0.31 to −1.2 MPa had no effect on controlling the level of vitrification. Gelrite promoted vitrification but GIBCO agar, alone or in combination with Gelrite, reduced its occurrence. Lowering media NH₄ content reduced vitrification, whereas sealing culture vessels with parafilm increased it. As it is now possible to control normal and vitreous plant development inPetunia, this can be used as a model system for studying the physiology and biochemistry of this developmental abnormality.     

Effect of ammonium-N on malic enzyme and lipid production in Rhodotorula glutinis grown on monosodium glutamate wastewater

Rhodotorula glutinis, an oil producing strain, can utilize monosodium glutamate (MSG) wastewater as a raw material for lipid production. The effects of ammonium-N in the MSG wastewater (ammonium 15,000–25,000?mg/L, COD 30,000–50,000?mg/L) on cell growth, lipid accumulation and malic enzyme activity of R. glutinis have been studied. Four initial ammonium sulfate concentrations in the medium were set, which were 20, 60, 100, and 140?g/L. With an increase in the ammonium sulfate concentration, the uptake of ammonia nitrogen and lipid accumulation increased while the biomass decreased at 72?h. The maximum value of ammonia nitrogen consumption reached 5.77?g/L for an initial ammonium sulfate concentration of 140?g/L at 72?h. In addition, 60?g/L ammonium sulfate concentration may be an appropriate concentration for R. glutinis cultivation. The activity of the malic enzyme was measured and the results showed that there was a linear relationship between the intracellular lipid content and the total malic enzyme activity.     

真盐生植物盐角草对不同氮形态的响应

盐角草(Salicornia europaea L.)是一种喜盐植物,其最佳生长的实现需要200—400 mmol/L NaCl。为了解盐渍环境下盐角草氮素吸收利用特点,在水培添加200或400 mmol/L NaCl情况下,从生长指标,光合参数,根系体积和活力,硝酸还原酶与谷氨酰胺合成酶活力,蛋白、总氮、硝态氮及铵态氮含量等方面检测了硝态氮、铵态氮和尿素3种氮形态对盐角草生长的影响。研究发现以氮摩尔浓度(mmol/L)计,在0.1—400 mmol/L浓度范围进行测试,盐角草在0.1 mmol/L低氮条件下仍能维持生长,同时,抑制盐角草生长的氮浓度域值较高,其中铵态氮、尿素和硝态氮分别为50、50及400 mmol/L。研究结果表明盐角草吸收利用氮素的能力强,对氮素的浓度耐受范围宽,3种氮形态都可作为氮源满足其生长需要,但有效促进生长的效果存在差异,总体顺序从高到低依次为硝态氮、铵态氮和尿素。研究为揭示盐生植物氮吸收利用特点提供了基础数据,对提高盐生植物生产力,指导沿海滩涂生态建设具有一定的指导意义。     

Studies on Pectic Enzymes of Microorganisms

Conditions for the production of endo-polygalacturonase (endo-PG) with Aspergillus saitoi IAM 2217 in the submerged culture was examined. This strain was selected as the most potent producer of endo-PG. Endo-PG of this strain was produced in the absence of pectin, but the addition of pectin increased endo-PG activity when inoculated with proliferated mycelia.

As far as examined with a modified Czapek medium (ordinary constituents + pectin and ammonium tartrate), the addition of organic nitrogen sources, such as corn steep liquor, markedly reduced the enzyme producibility. As for the carbon and nitrogen amount in the medium, sucrose: 4%, pectin: 2%, NaNO₃: 1.15%, C/N = 10, gave the best result among tested.     

Lysine catabolism inStreptomyces ambofaciens producer of macrolide antibiotic,spiramycin

Spiramycin production was highly stimulated when lysine was used as the sole nitrogen source. This amino acid was catabolized by the -transaminase pathway characterized by dosage of cadaverine aminotransferase (CAT) enzyme. The Km_cadaverine was of 57mM. CAT was highly induced by lysine (634% in comparison with ammonium). Addition of 40mm of ammonium in a culture begun with 20mm of lysine as the sole initial nitrogen source repressed CAT biosynthesis by 24% but did not affect spiramycin production seriously. Addition of 20mm of lysine in a culture started with 40mm ammonium induced CAT biosynthesis of 425%, but did not allow spiramycin production. In these two cases, spiramycin production seems to be conditioned by the nitrogen source initially present in the culture medium. CAT activity was inhibited by ammonium ions (33% at 20mm), whereas lysine had no effects.     

Effect of levulinic acid on pigment biosynthesis in Agmenellum quadruplicatum.

When levulinic acid was added to a growing culture of the cyanobacterium (blue-green alga) Agmenellum quadruplicatum PR-6, delta-aminoelevulinic acid accumulated in the medium and chlorophyll a synthesis and cell growth were inhibited, but there was a small amount of c-phycocyanin synthesis. The amount of delta-aminolevulinic acid produced in the treated culture did not fully account for the amount of pigment synthesized in the untreated control. Levulinic acid and either sodium nitrate or ammonium chloride were added to nitrogen-starved cultures of PR-6, and delta-aminolevulinic acid production and chlorophyll a and c-phycocyanin content were monitored. When ammonium chloride was added as a nitrogen source after nitrogen starvation, the cells recovered more rapidly than when sodium nitrate was added as a nitrogen source. In cultures recovering from nitrogen starvation, synthesis of c-phycocyanin occurred before synthesis of chlorophyll a.     

Kinetic properties and ammonium-dependent regulation of cytosolic isoenzymes of glutamine synthetase in Arabidopsis

Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme of nitrogen assimilation, catalyzing the synthesis of glutamine from ammonium and glutamate. In Arabidopsis, cytosolic GS (GS1) was accumulated in roots when plants were excessively supplied with ammonium; however, the GS activity was controlled at a constant level. The discrepancy between the protein content and enzyme activity of GS1 was attributable to the kinetic properties and expression of four distinct isoenzymes encoded by GLN1;1, GLN1;2, GLN1;3 and GLN1;4, genes that function complementary to each other in Arabidopsis roots. GLN1;2 was the only isoenzyme significantly up-regulated by ammonium, which correlated with the rapid increase in total GS1 protein. GLN1;2 was localized in the vasculature and exhibited low affinities to ammonium (Km = 2450 +/- 150 microm) and glutamate (Km = 3.8 +/- 0.2 mm). The expression of the counterpart vascular tissue-localizing low affinity isoenzyme, GLN1;3, was not stimulated by ammonium; however, the enzyme activity of GLN1;3 was significantly inhibited by a high concentration of glutamate. By contrast, the high affinity isoenzyme, GLN1;1 (Km for ammonium < 10 microm; Km for glutamate = 1.1 +/- 0.4 mm) was abundantly accumulated in the surface layers of roots during nitrogen limitation and was down-regulated by ammonium excess. GLN1;4 was another high affinity-type GS1 expressed in nitrogen-starved plants but was 10-fold less abundant than GLN1;1. These results suggested that dynamic regulations of high and low affinity GS1 isoenzymes at the levels of mRNA and enzyme activities are dependent on nitrogen availabilities and may contribute to the homeostatic control of glutamine synthesis in Arabidopsis roots.     

Regulation of glutamine metabolism in Chlorella pyrenoidosa. Regulation of glutamine synthetase activity by adenylic system components]

A decrease of glutamine synthetase (E. C. 6.3.1.2.) activity was observed under the assimilation of ammonium nitrogen in Chlorella. At the same time a decrease of ATP content in Chlorella cells took place. The ATP content was 7-fold decreased, while ADP and AMP contents were 4-fold and 3-fold increased respectively, after 15 min. of Chlorella incubation on "ammonium" medium. Further incubation for 45 min, resulted in gradual increase of ATP content and in decrease of ADP and AMP contents. The value of energy charge in ammonium assimilating Chlorella cells sharply decreased for first 15 min. of incubation and then it normalized gradually. The experiments with glutamine synthetase preparation, isolated from ammonium assimilating cells, have shown that ADP and AMP are strong inhibitors of the enzyme in the presence of Mg2+, and only ADP produces the inhibitory effect in the presence of Mn2+. No enzyme reactivation was observed after the transfer of ammonium assimilating cells into nitrogen-free medium or nitrate medium, the enzyme activity increasing at the expense of enzyme protein synthesis denovo.     

Ascorbic Acid and Heterocyst Development in the Blue–Green Alga Amabaema ambigua

The nitrogen–fixing blue–green alga Anabaena ambigua was grown in a medium which contained either ammonium chloride as nitrogen source or molecular nitrogen. In the latter case the alga produced heterocysts. The material was analysed for ascorbic acid, dehydro-ascorbic acid and diketogulonic acid. The amount of a,scorbic acid was found always to he higher in the alga grown with molecular nitrogen. When the alga grown with combined nitrogen was transferred to the medium lacking it, there was an increase in the ascorbic acid content. Conversely, when material cultured on the nitrogen–free medium was suspended in the medium containing ammonium chloride, there was a decrease in the cellular ascorbic acid. Esogenously added ascorbic acid, up to 0.5 mg per ml, increased the heterocyst frequency to nearly three times that of the control. D–isoascorbic acid, an analogue of ascorbic acid, also showed an enhancement of heterocyst production. Algal extracts were fractionated by poiyacrylamide electro–phoresis, and the presence of ascorbic acid oxidase was detected on the gels. Two bands, with Rf values 0.34 and 1.0, were found to give positive test: for the enzyme. The total enzyme activity was 16.7 % higher in cells grown with molecular nitrogen than in those grown with combined nitrogen. The exact location of the enzyme in the alga ist not known although the heterocysts were earlier shown to contain ascorbic acid. Cytochemical tests, however, indicated strong per–oxidase activity in the heterocysts.     

Nitrogen Incorporation into Lignite Humic Acids during Microbial Degradation

Previous study showed that nitrogen content in lignite humic acids (HA) increased significantly during lignite biodegradation. In this paper we evaluated the factors responsible for the increased level of N in HA and the formation of new nitrogen compound following microbial degradation. When the ammonium sulfate concentration in lignite medium was < 0.5%, the N-content in HA was higher than that in the crude lignite humic acid (cHA); when the ammonium sulfate concentration was ≥ 0.5%, both the biodegraded humic acid (bHA) N-content and the content of bHA in lignite increased significantly, but at 2.0% no increase was observed. This indicated that HA incorporated N existing in the lignite medium, and more HA can incorporate more N with the increase of bHA amount in lignite during microbial degradation. CP/MAS ¹⁵N NMR analysis showed that the N incorporated into HA during biotransformation was in the form of free or ionized NH₂-groups in amino acids and sugars, as well as NH₄ ⁺. We propose nitrogen can be incorporated into HA biotically and abiotically. The high N content bHA has a potential application in agriculture since N is essential for plant growth.     

Production, purification, and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3

A thermophilic isolate Bacillus coagulans BTS-3 produced an extracellular alkaline lipase, the production of which was substantially enhanced when the type of carbon source, nitrogen source, and the initial pH of culture medium were consecutively optimized. Lipase activity 1.16 U/ml of culture medium was obtained in 48 h at 55 degrees C and pH 8.5 with refined mustard oil as carbon source and a combination of peptone and yeast extract (1:1) as nitrogen sources. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The enzyme showed maximum activity at 55 degrees C and pH 8.5, and was stable between pH 8.0 and 10.5 and at temperatures up to 70 degrees C. The enzyme was found to be inhibited by Al3+, Co2+, Mn2+, and Zn2+ ions while K+, Fe3+, Hg2+, and Mg2+ ions enhanced the enzyme activity; Na+ ions have no effect on enzyme activity. The purified lipase showed a variable specificity/hydrolytic activity towards various 4-nitrophenyl esters.     

绿僵菌产海藻糖水解酶培养条件研究

丝状真菌绿僵菌能产生一系列二糖水解酶,其中包括海藻糖水解酶。这些酶在绿僵菌对昆虫的致病过程中起着重要的作用。本文研究了不同碳源、氮源对金龟子绿僵菌Metarhizium anisopliae var. acridum菌株CQMa102产生与分解昆虫血淋巴中海藻糖等二糖相关的海藻糖水解酶活性的影响。结果表明:分别以葡萄糖、麦芽糖、蔗糖、山梨醇和可溶性淀粉为碳源,金龟子绿僵菌均可产生海藻糖水解酶,但最佳碳源是可溶性淀粉,因为由其诱导产生的海藻糖水解酶具有最高的总活性和比活性以及更多的同工酶,山梨醇次之。硝态氮(NaNO₃)作为唯一氮源时,几乎检测不出海藻糖水解酶活性,而铵态氮((NH₄)₂SO₄)或NaNO₃和有机氮(蛋白胨和酵母浸膏)混合氮源作氮源时,海藻糖水解酶活性都很高。在绿僵菌菌丝提取液和滤液的海藻糖水解酶活性比较中发现:CQMa102在多数碳源的培养基中产生的海藻糖水解酶主要分泌到培养基中,仅有少数结合在细胞壁上。     

Isolation and characterization of xanthine dehydrogenase from Chlamydomonas reinhardtii

Xanthine dehydrogenase (XDH, EC 1.2.1.37) of Chlamydomonas reinhardtii (Sager) 6145c wild strain has been isolated and characterized for the first time in a unicellular green alga. The enzyme has an M_r of 330 kDa, and FAD, molybdenum and iron are cofactors required for its activity as deduced from results obtained using specific inhibitors, ⁵⁹Fe-labelling experiments, activity protection by FAD, physiological responses in vivo to iron and molybdenum deficiencies in the culture medium and work with mutants lacking molybdenum cofactor. Xanthine dehydrogenase exhibited Mi-chaelian kinetics typical for a bisubstrate enzyme with apparent K_m values for NAD ⁺, hypoxanthine and xanthine of 35, 160 and 70 μ M , respectively. Under phototrophic conditions enzyme activity was repressed by ammonium, but xanthine was not required for the enzyme to be induced, since high levels of enzyme activity were found in cells grown on ammonium and transferred to either N-frec media or media containing either of the nitrogen sources adenine, urea, urate, xanthine, hypoxanthine and guanine.     

Purification, crystallization, and characterization of peroxidase from Coprinus cinereus

Peroxidase (donor: H2O2 oxidoreductase [EC 1.11.1.7]) was purified from a culture broth of an inkcap Basidiomycete, Coprinus cinereus S.F. Gray. A single component containing a low amount of carbohydrate was isolated by affinity chromatography on concanavalin A-Sepharose and crystallized from ammonium sulfate solution. The enzyme is an acidic protein (pI 3.5) and consists of a single polypeptide chain having the molecular weight of 41,600 daltons. The enzyme contains one protohemin per molecule and exhibits the characteristic absorption, circular dichroism, and magnetic circular dichroism spectra of a heme-protein. The Coprinus peroxidase forms two characteristic intermediate compounds, I and II, and the rate constants for hydrogen peroxide and guaiacol had similar values to those for higher plant peroxidases. The ferric enzyme formed a cyanide compound with a dissociation constant similar to those for higher plant enzyme, but the dissociation constant of the ferrous enzyme-cyanide was large. The chemical composition of Coprinus peroxidase showed 381 amino acid residues, 1 glucosamine, 3 true sugars, 3 calcium, and 1 non-heme iron other than 1 protohemin. The secondary structure of the fungal enzyme was very similar to that of horseradish peroxidase.     

Effect of nitrogen starvation on ammonium-inhibition of nitrogenase activity in Azotobacter chroococcum

When Azotobacter chroococcum cells grown in batch culture under N₂-fixing conditions were transferred to a medium lacking a nitrogen source, the cellular C/N ratio, the amount of alginic acid released into the external medium and the rate of endogenous respiration increased appreciably after 6 h to the exclusion of dinitrogen, whereas nitrogenase activity did not undergo any significant change. Nitrogen deficiency caused a decrease in the ammonium inhibition of nitrogenase activity from 95% inhibition at zero time to 14% after 6 h incubation under dinitrogen starvation, with no difference in the rate of ammonium utilization by N₂-fixing and N₂-starved cells being observed. This suggests that a balance of nitrogen and carbon assimilation is necessary for the ammonium inhibition of nitrogenase activity in A. chroococcum to take place.     

How does glutamine synthetase activity determine plant tolerance to ammonium?

The wide range of plant responses to ammonium nutrition can be used to study the way ammonium interferes with plant metabolism and to assess some characteristics related with ammonium tolerance by plants. In this work we investigated the hypothesis of plant tolerance to ammonium being related with the plants’ capacity to maintain high levels of inorganic nitrogen assimilation in the roots. Plants of several species (Spinacia oleracea L., Lycopersicon esculentum L., Lactuca sativa L., Pisum sativum L. and Lupinus albus L.) were grown in the presence of distinct concentrations (0.5, 1.5, 3 and 6 mM) of nitrate and ammonium. The relative contributions of the activity of the key enzymes glutamine synthetase (GS; under light and dark conditions) and glutamate dehydrogenase (GDH) were determined. The main plant organs of nitrogen assimilation (root or shoot) to plant tolerance to ammonium were assessed. The results show that only plants that are able to maintain high levels of GS activity in the dark (either in leaves or in roots) and high root GDH activities accumulate equal amounts of biomass independently of the nitrogen source available to the root medium and thus are ammonium tolerant. Plant species with high GS activities in the dark coincide with those displaying a high capacity for nitrogen metabolism in the roots. Therefore, the main location of nitrogen metabolism (shoots or roots) and the levels of GS activity in the dark are an important strategy for plant ammonium tolerance. The relative contribution of each of these parameters to species tolerance to ammonium is assessed. The efficient sequestration of ammonium in roots, presumably in the vacuoles, is considered as an additional mechanism contributing to plant tolerance to ammonium nutrition.