On the specificity of alcoholic acidic silver nitrate reagent for the histochemical localization of ascorbic acid

Summary The specificity of the alcoholic acidic silver nitrate staining method for the histochemical localization of ascorbic acid was reappraised. It was found that the method is by and large better suited for the localization of ascorbic acid in both animal and plant tissues due to its greater specificity, which is ensured by employing reagent made in carbon dioxide saturated glass distilled water as well as by carrying out the reaction at a low temperature (0–4° C) and at a pH of 2–2.5.Paper presented at the 65th Session, Indian Science Congress, Ahmedabad, Jan. 3–7, 1978     

Content of Oxalate in Actinidia Deliciosa Plants Grown in Nutrient Solutions with Different Nitrogen Forms

Kiwifruit plants (Actinidia deliciosa cv. Hayward) were grown in Hoagland nutrient solution with calcium nitrate, potassium nitrate, ammonium nitrate or ammonium chloride as the nitrogen source. Plants grown in the solution with nitrate nitrogen displayed a higher oxalate content, greater shoot length and leaf area, and higher content of ascorbic acid and NO₃ ^– ions in the leaves. Plants grown in the solution with ammonium nitrate, and particularly with ammonium chloride, showed low oxalate content, low content of ascorbic acid and NO₃ ^–, high content of Cl^– and Na⁺, low shoot length and leaf area. Oxalate formation appeared to be connected with the assimulation of nitrate, more precisely with nitrate reduction, while ammonium nitrogen assimilation did not induce the synthesis of oxalic acid.     

Modulation of mango ripening by chemicals: physiological and biochemical aspects

During ripening, fleshy fruits undergo textural changes that lead to loss of tissue firmness and consequent softening due to cell wall dismantling carried out by different and specifically expressed enzymes. The effect of various chemical treatments on the ripening of mango fruit (Mangifera indica) was investigated at physiological and biochemical level. Based on changes in respiration, firmness, pH, total soluble sugar and a cell wall degrading enzyme pectate lyase (PEL) activity, treatment with 1-methylcyclopropene (1-MCP), silver nitrate (AgNO₃), gibberlic acid (GA₃), sodium metabisulphite (SMS) and ascorbic acid led to delaying of ripening process while those of ethrel and calcium chloride (CaCl₂) enhanced the process. PEL of mango was found to be inhibited by certain metabolites present in dialysed ammonium sulphate enzyme extract as well as EDTA. Mango PEL activity exhibited an absolute requirement for Ca²⁺ and an optimum pH of 8.5.     

Is alcoholic acidic silver nitrate reagent really specific for the histochemical localization of ascorbic acid?

Summary The histochemical localization of ascorbic acid in plant tissues with the alcoholic acidic silver nitrate reagent is shown here to be not specific for ascorbic acid, since some of the polyphenolic substances, including flavonoids, which are known to be widely distributed in plant tissues, are also able to reduce the acidic alcoholic silver nitrate reagent at low temperature (0–4°C) and at pH 2 to 2.5 in dark. This method may perhaps be used for animal tissues where flavonoid pigments do not occur in such large quantities as they do in plants. I therefore, come to the inevitable conclusion that the use of alcoholic acidic silver nitrate reagent in localizing ascorbic acid in plant tissues may be highly misleading.     

Effect of silver nitrate on multiple shoot formation of Virginia-type peanut through shoot tip culture

Summary A protocol for micropropagation of Virginia-type peanut plants, an ancient crop of the New World, is reported. This study was conducted to explore the effect of silver nitrate (AgNO₃), alone or in combination with growth regulators, on multiple shoot formation from shoot tip culture. Incorporation of AgNO₃ into the medium, without growth regulators, induced regeneration of the explants (which did not develop at all in the AgNO₃-free medium), and stimulated the emergence of axillary shoots. When AgNO₃ was added in combination with cytokinins and α-naphthaleneacetic acid (NAA), maximum average shoot number per regenerating explant was recorded (6.3) in Murashige and Skoog (MS) medium containing 33 μM 6-benzyladenine, 5.3 μM NAA, and 23.54 μM AgNO₃. Moreover, AgNO₃ showed a positive and marked effect on both shoot elongation and the reduction of callus proliferation from the basal ends of shoot tips. Following a period of elongation, the shoots were rooted in hormone-free Ms medium, showing no residual effects due to the long-term culture in AgNO₃-containing media. Acclimatization was easily obtained after plantlets were transferred to pots under greenhouse conditions, with 90% survival.     

The effects of silver nitrate and different carbohydrate sources on somatic embryogenesis in Coffea canephora

The response of five Coffea canephora Pierre genotypes with regard to somatic embryogenesis was tested on media containing silver nitrate (AgNO₃) and different carbohydrates (sucrose, fructose, maltose and glucose). The presence of AgNO₃ caused only small modifications to the ionic equilibrium of the media. At concentrations between 30–60 M, AgNO₃ improved embryo yield for the genotypes evaluated, while higher doses negatively affected the regenerative capacity. The substitution of maltose, glucose or fructose for sucrose produced different responses depending on the genotype. Fructose significantly increased somatic embryo production in genotypes N91 and N128, while maltose was highly effective for N75. In addition, more synchronous embryo development was observed in genotype N91 when glucose was used instead of sucrose.     

Is alcoholic acidic silver nitrate reagent really specific for the histochemical localization of ascorbic acid.

The histochemical localization of ascorbic acid in plant tissues with the alcoholic acidic silver nitrate reagent is shown here to be not specific for ascorbic acid, since some of the polyphenolic substances, including flavonoids, which are known to be widely distributed in plant tissues, are also able to reduce the acidic alcoholic silver nitrate reagent at low temperature (0-4 degrees C) and at pH 2 to 2.5 in dark. This method may perhaps be used for animal tissues where flavonoid pigments do not occur in such large quantities as they do in plants. I therefore, come to the inevitable conclusion that the use of alcoholic acidic silver nitrate reagent in localizing ascorbic acid in plant tissues may be highly misleading.     

Purification and characterization of chitinase from the stomach of silver croaker Pennahia argentatus

A chitinase was purified from the stomach of a fish, the silver croaker Pennahia argentatus, by ammonium sulfate fractionation and column chromatography using Chitopearl Basic BL-03, CM-Toyopearl 650S, and Butyl-Toyopearl 650S. The molecular mass and isoelectric point were estimated at 42 kDa and 6.7, respectively. The N-terminal amino acid sequence showed a high level of homology with family 18 chitinases. The optimum pH of silver croaker chitinase toward p-nitrophenyl N-acetylchitobioside (pNp-(GlcNAc)₂) and colloidal chitin were observed to be pH 2.5 and 4.0, respectively, while chitinase activity increased about 1.5- to 3-fold with the presence of NaCl. N-Acetylchitooligosaccharide ((GlcNAc)_n, n = 2–6) hydrolysis products and their anomer formation ratios were analyzed by HPLC using a TSK-GEL Amide-80 column. Since the silver croaker chitinase hydrolyzed (GlcNAc)_4–6 and produced (GlcNAc)_2–4, it was judged to be an endo-type chitinase. Meanwhile, an increase in β-anomers was recognized in the hydrolysis products, the same as with family 18 chitinases. This enzyme hydrolyzed (GlcNAc)₅ to produce (GlcNAc)₂ (79.2%) and (GlcNAc)₃ (20.8%). Chitinase activity towards various substrates in the order pNp-(GlcNAc)_n (n = 2–4) was pNp-(GlcNAc)₂ >> pNp-(GlcNAc)₄ > pNp-(GlcNAc)₃. From these results, silver croaker chitinase was judged to be an enzyme that preferentially hydrolyzes the 2nd glycosidic link from the non-reducing end of (GlcNAc)_n. The chitinase also showed wide substrate specificity for degrading α-chitin of shrimp and crab shell and β-chitin of squid pen. This coincides well with the feeding habit of the silver croaker, which feeds mainly on these animals.     

Structural characterizations of some adducts of silver(I) nitrate and perchlorate with some cyclic di- (or tri-) ene organic ligands

Single crystal X-ray structural characterizations of some adducts of silver(I) nitrate and perchlorate with assorted organic poly-ene ligands (nbd = norbornadiene, bicyclo[2.2.1]hepta-2,5-diene; cod = 1,5-cyclooctadiene; cdt ≡ trans,trans,cis-cyclododeca-1,5,9-triene) are reported, all being polymeric in form (with the exception of mononuclear ionic AgClO₄:cod (1:2)), with chains comprised of alternating silver and nitrate/perchlorate components substituted or linked by unsaturated donors which complete the coordination spheres of the silver atoms. In AgNO₃:nbd (2:1) (a redetermination), pairs of silver/nitrate strands are linked in a one-dimensional polymer by the nbd ligands. In AgNO₃:nbd (1:1)_∞, meandering silver/nitrate strands containing pairs of independent silver and nitrate units in a crystallographic mirror plane are linked to either side with parallel planes by nbd ligands. In AgNO₃:cod (1:1)_∞, the cod ligands ‘chelate’ to the silver atoms in a silver/nitrate chain. In AgNO₃:cdt (1:2)_∞, pairs of ‘unidentate’ cdt ligands are pendant from a silver/nitrate chain, while in the (1:1)_∞ adduct, the cdt ligands bridge pairs of silver atoms from an adjacent chain forming a two-dimensional web. A common form of the bridging nitrate group in the above is as an O,O′-NO₃-O′,O″ bis-chelate, the pair of the bis-oxygen chelates having a common oxygen atom.     

AN ELECTRON MICROSCOPE STUDY OF SILVER NITRATE REDUCTION IN LEAF CELLS

Brown , W. V., H. Mollenhauer , and C. Johnson . (U. Texas, Austin.) An electron microscope study of silver nitrate reduction in leaf cells. Amer. Jour. Bot. 49(1): 57–63. Illus. 1962.—As reported earlier in many studies, AgNO₃ is reduced quickly by the living chloroplasts of angiosperms. Electron microscope study has resolved the conflict of opinions concerning the exact location of the silver particles. Reduction of AgNO₃, as indicated by location of silver particles, occurs within the chloroplasts but not within the grana or pure stroma; it appears to be associated with the intergranal (also called stroma) lamellae. Silver particles are formed also at the surfaces of the cell wall, both in the middle lamella and at the inner surface, and also within plasmodesmata. It is concluded that chlorophyll is probably not involved directly in the reduction. There is some slight support for the popular hypothesis that ascorbic acid may be the chief reducing agent.     

Silver Ions Increase Auxin Efflux Independently of Effects on Ethylene Response

Silver nitrate and aminoethoxyvinylglycine (AVG) are often used to inhibit perception and biosynthesis, respectively, of the phytohormone ethylene. In the course of exploring the genetic basis of the extensive interactions between ethylene and auxin, we compared the effects of silver nitrate (AgNO₃) and AVG on auxin responsiveness. We found that although AgNO₃ dramatically decreased root indole-3-acetic acid (IAA) responsiveness in inhibition of root elongation, promotion of DR5-β-glucuronidase activity, and reduction of Aux/IAA protein levels, AVG had more mild effects. Moreover, we found that that silver ions, but not AVG, enhanced IAA efflux similarly in root tips of both the wild type and mutants with blocked ethylene responses, indicating that this enhancement was independent of ethylene signaling. Our results suggest that the promotion of IAA efflux by silver ions is independent of the effects of silver ions on ethylene perception. Although the molecular details of this enhancement remain unknown, our finding that silver ions can promote IAA efflux in addition to blocking ethylene signaling suggest that caution is warranted in interpreting studies using AgNO₃ to block ethylene signaling in roots.     

Synergistic effect of ethylene inhibitors and putrescine on shoot regeneration from hypocotyl explants of Chinese radish (Raphanus sativus L. var. longipinnatus Bailey) in vitro

Summary The role of ethylene and putrescine on shoot regeneration from hypocotyl explants of Chinese radish (Raphanus sativus L. var. longipinnatus Bailey cv. Red Coat) was investigated. Explants were recalcitrant in culture, but exogenous application of ethylene inhibitor [20–30 M aminoethoxyvinylglycine (AVG) or AgNO₃] enhanced shoot regeneration of explants grown on medium supplemented with 2 mg/l N⁶-benzyladenine and 1 mg/l 1-naphthaleneacetic acid. The best regeneration occurred in the medium containing AgNO₃ in combination with AVG. Culture medium solidified with agarose in the presence of AgNO₃ but not AVG was also beneficial to shoot regeneration. Exogenous putrescine, 2-chloroethylphosphonic acid and 1-aminocyclopropane-1-carboxylate had no effect on shoot regeneration. However, regeneration was greatly promoted by 10–25 mM putrescine in combination with 30 M AgNO₃ or AVG. Explants with high regenerability grown in the presence of AgNO₃ or in combination with putrescine emanated high levels of ethylene throughout the 21-d culture period. By contrast, AVG or putrescine alone resulted in a decrease in ethylene production. For rooting of shoot cuttings, IAA and IBA at 1–5 mg/l were more effective than NAA.Abbreviations ACC 1-aminocyclopropane-1-carboxylate - AVG aminoethoxyvinylglycine - BA N⁶-benzyladenine - CEPA 2-chloroethylphosphonic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PAs polyamines - SAM S-adenosyl-L-methionine     

Chemical evolution of dehydrogenases: Amino acid pentacyanoferrate (II) as possible intermediates

Dehydrogenation of ascorbic acid and reduced nicotinamide adenine dinucleotide (NADH) with methylene blue using complexes of the type [Fe(II)(CN)₅ (L)] ^n– (wheren=3 or 4; L=glycine, histidine, imidazole, and triglycine) as catalyst have been studied at pH 9.18. Similar kinetic behavior was observed for the dehydrogenation of ascorbic acid as well as for NADH; both reactions showed first order dependency on the substrates. First order dependence was observed only at lower concentrations of methylene blue; at higher concentrations of methylene blue, the reactions were independent of methylene blue. The order with respect to catalyst varied between 0.3–0.5. A tentative mechanism which conforms to the observed kinetics has been proposed. It is believed that on the primitive earth when the reducing potential of the atmosphere was not high enough, lower oxidation state iron complexes like [Fe(II)(CN)₅(L)] ^n– might have been involved in dehydrogenase-type activity.     

The permeability of the epidermal cell plasma membrane of barley leaves to abscisic acid

Uptake of ³H-labelled (±)-abscisic acid (ABA) into isolated barley (Hordeum vulgare L.) epidermal cell protoplasts (ECP) was followed over a range of pH values and ABA concentrations. The present results show that ABA uptake is not always linearly correlated with the external concentration of undissociated ABA (ABAH). At pH 7.25, ABA uptake exhibited saturation kinetics with an apparent K _m value of 75 mmol·m^–3 to tal ABA. This saturable transport component was inhibited by pretreating the protoplasts with 1 mol·m^–3 p-chloromercuribenzenesulfonic acid at pH 8.0, conditions that minimized the uptake of this acid sulfhydryl reagent. Moreover, the rate of (±)-[^3]HABA uptake was reduced by addition of 0.1 mol·m^–3 (±)-ABA to 41%, whereas the same concentration of (±)-ABA was approximately half as effective (46% of the inhibitory effect). Thus, it was concluded that only (±)-ABA competes for an ABA carrier that is located in the epidermal cell plasma membrane. The permeability of the epidermal cell plasma membrane was studied by performing a Collander analysis. At pH 6 the overall plasma-membrane permeability of epidermal cells was similar to that of guard cells but was about two times higher than that of mesophyll cells.Abbreviations ABA abscisic acid - ABA^– anion of ABA - ABAH undissociated ABA - 2,4-D 2,4-dichlorophenoxyacetic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - ECP deepidermal cell protoplast - K_r partition coefficient - M_r relative molecular mass - NEM N-ethylmaleimide - PCMBS p-chloromercuriben zenesulfonic acid - P_s permeability coefficient We are grateful to Barbara Dierich for expert technical assistance, to Prof. H. Gimmler (Lehrstuhl für Botanik I, Universität Würzburg, FRG) for helpful discussions and to the Deutsche Forschungsgemeinschaft (SFB 251, TP 3) for financial support.     

Interaction of nitrate uptake and nitrogen fixation in faba beans

An experiment was carried out to determine the relationship between nitrate uptake and nitrogen fixation of faba beans. Therefore inoculated and uninoculated faba beans were grown in nutrient solution with different nitrate concentrations. Nitrate uptake was measured every two days during the growing period. At the end of the experiment the nitrate uptake kinetics were determined with a short time depletion technique and nitrogen fixation was measured with the acetylene reduction method. A limitation of nitrate uptake due to nitrogen fixation was relatively small. Nitrate concentrations of approximately 1 mol m^–3 and 5 mol m^–3 decreased nitrogen fixation to values of 16% and 1% of the control plants which received no nitrate nitrogen. A reduction of nitrogen fixation was mainly due to a decrease of specific nitrogen fixation per unit nodule weight and to a lesser extent due to a reduction of nodule growth. Only the maximum nitrate influx (I_max) seemed to be influenced by nitrogen fixation. Michaelis-Menten constants (K_m) and minimum NO _inf3 ^– -concentrations (C_min) were not significantly influenced by nitrogen fixation.     

Effects of low nitrogen medium on endogenous changes in ethylene, auxins, and cytokinins in in vitro shoot formation from rhizomes of Cymbidium kanran

Summary Shoot formation from rhizome explants of Cymbidium kanran was promoted on Murashige and Skoog (MS) medium: (1) with 1 mgl⁻¹ (4.4μM) 6-benzyladenine (BA) and 0.1 mgl⁻¹ (0.54μM) α-naphthaleneacetic acid (NAA); (2) with ethylene inhibitor (silver nitrate, AgNO₃); or (3) by reducing ammonium nitrate (NH₄NO₃) and potassium nitrate (KNO₃) to 25 and 50%, respectively, of their original concentrations. Shoot formation by BA and NAA was strongly inhibited with the application of ethephon, an ethylene releaser. The ethylene production from the rhizome explants was reduced 30–55% on low nitrogen medium after 1–3 mo. of culture and 52% on BA and NAA medium after 1 mo. of culture compared with explants on standard MS medium. No difference in endogenous auxin (indole-3-acetic acid, IAA) and cytokinin (isopentenyl adenosine, iPA) contents in the rhizomes was found between the treatments. Low ethylene levels were correlated with higher frequency of shoot formation from the rhizomes.     

Protective effect of seminal plasma proteins on the degradation of ascorbic acid

The objectives of this study were to determine ascorbic acid stability and its effect on antiproteinase activity of seminal plasma in the presence of an oxidant. Effect of seminal plasma, and additives: glutathione, albumin, hydrogen peroxide and Tris buffer, on ascorbic acid degradation was investigated by UV absorbance. Antiproteinase against trypsin amidase activity was measured spectrophotometrically using N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as substrate. Ascorbic acid was destroyed much more rapidly with the addition of hydrogen peroxide than in Tris buffer at pH 8.2 alone. Seminal plasma protected ascorbic acid more efficiently than glutathione and albumin alone. The protective effect of seminal plasma on ascorbic acid degradation may closely relate to the function of ascorbic acid in reproductive system of scurvy-prone animals including teleost fish. Within the range of 1–8 mM concentrations, ascorbic acid had a pro-oxidant action on seminal plasma antiproteinase activityin vitro when they were incubated with hydrogen peroxide.Abbreviations AA Ascorbic acid - BAPNA N-benzoyl-DL-arginine-p-nitroanilide - DMSO dimethyl sulfoxide - GSH glutathione - H₂O₂ hydrogen peroxide     

Stimulation of shoot regeneration from cotyledons of Helianthus annuus by the ethylene inhibitors,silver and cobalt

The effects of CoCl₂, AgNO₃ and ethylene released by exogenous 2-chloroethylphosphonic acid (Ethephon), were studied on shoot regeneration from cotyledons of Helianthus annuus cv. E8206R, a poorly regenerative cultivar. Inhibition of ethylene biosynthesis by CoCl₂, at concentrations of 20 K, provoked a substantial enhancement of shoot regeneration (30 %): the control was poorly regenerative. However, CoCl₂ had no effect when Ethephon was supplied. Inhibition of ethylene action by AgNO₃, at concentrations of 10–25 M, caused a significant increase in plant regeneration: 25 % instead of 1.2 % in the control. Furthermore, addition of Ethephon to AgNO₃-treated tissues failed to reduce the stimulation of shoot regeneration caused by AgNO₃. On the basis of these findings, it is suggested that ethylene inhibits the regeneration process from cotyledons of sunflower.Abbreviations NAA 1-naphthalene acetic acid - BAP 6-benzylamino-purine - GA₃ gibberellic acid - Ethephon 2-chloroethylphosphonic acid - MS Murashige and Skoog medium - AVG aminoethoxyvinylglycine     

Ascorbic acid promotes prostanoid release in human lung parenchyma

Ascorbic acid reduces airway reactivity to inhaled bronchoconstrictor agents in man and guinea pigs. The precise mechanism(s) responsible for this effect are unknown, but in both species an acute indomethacin treatment reverses the action of the ascorbic acid. To determine if ascorbic acid promotes prostanoid synthesis and/or inhibits degradation, human lung parenchymal slices (100–200mg) were incubated for 60 minutes in oxygenated Tyrode's solution alone or with sodium ascorbate (0.001M–1M) and/or methacholine (1μM–100μM) and/or indomethacin (0.17μM–17μM). Aliquots of the incubation medium were assayed by radioimmunoassay for PGE₂, PGF_2α, thromboxane B₂ and 6-keto-PGF_1α. Ascorbic acid increased the accumulation of all four prostanoids in the incubation medium, especially thromboxane B₂ and 6-keto-PGF_1α. This stimulatory effect of ascorbic acid was concentration-dependent and was inhibited by indomethacin. We conclude that ascorbic acid can alter prostanoid generation by human lung tissue and this effect may, in part, explain its antibronchoconstrictor activity in man.