Analysis of variability in the amaranthus betacyanin assay for cytokinins: effects of "aging" excised cotyledons

“Aging” of excised cotyledons plus the top part of the hypocotyl of Amaranthus tricolor seedlings was carried out by washing in distilled H₂O for varying periods. This led to increased betacyanin accumulation during the subsequent 24-hour induction period in the presence of tyrosine and Na⁺ + K⁺ phosphate. Endogenous accumulation as well as that dependent on added benzyladenine and on added fusicoccin was stimulated. This stimulation could not be due to a carryover of a wound-induced burst of ethylene since 2-chloroethylphosphonic acid (Ethrel) was shown to be extremely inhibitory to betacyanin synthesis if present during the induction process. It is possible that a wound-induced burst of ethylene could give rise to increased betacyanin synthesis as an after effect. The procedure for obtaining good induction with the most reproducible results is described.     

Evidence against an involvement of cyclic nucleotides in the induction of betacyanin synthesis by cytokinins.

1. A wide range of purine bases, nucleosides and cyclic nucleotides were shown to induce betacyanin synthesis in Amaranthus seedlings. 2. The induction of pigment by benzyladenine, dibutyryl cyclic AMP or cyclic AMP was not potentiated by aminophylline. Aminophylline was shown to inhibit Amaranthus cyclic AMP phosphodiesterase activity. 4. Incubation of seedlings with aminophylline inhibited the conversion of 6-[G--3H]benzyladenine into presumed 9- and 7-glucosylbenzyladenine. 5. Induction of betacyanin synthesis by 6-benzyladenine or by exposure to red light was not accompanied by changes in the total cyclic AMP content in seedlings. 6. It is concluded that the inducers tested act as cytokinin analogues; no evidence was obtained to support cyclic AMP as an intermediate in the induction process.     

The pathway of betalain biosynthesis: Effect of cytokinin on enzymic oxidation and hydroxylation of tyrosine in Amaranthus tricolor seedlings

The effect of exogenously added tyrosine or l -3-(3,4-dihydroxyphenyl) alanine on the accumulation of betacyanin in response to cytokinin in Amaranthus tricolor (L.) var. tricolor half-seedlings depends on the age of the seedlings and the treatment of the seedlings prior to induction of pigment by benzyladenine. Neither extracted polyphenol oxidase, peroxidase or tyrosine hydroxylase activity, nor in vivo tyrosine hydroxylation is increased in response to exposure of seedlings to cytokinin. However a small percentage of the polyphenol oxidase activated or unmasked by Triton X-100 treatment of membrane fractions is increased after cytokinin treatment of half-seedlings for 22 h. It is concluded that cytokinin control may be on a multi-enzyme membrane-located complex involving part of the polyphenol oxidase activity of the tissue (possibly an isoenzyme), and that the majority of the polyphenol oxidase activity in Amaranthus is a constitutive membrane enzyme which is not involved in betacyanin synthesis. Although cytokinins do not affect in vivo tyrosine hydroxylation, this activity follows closely the accumulation of betacyanin which is first detectable about 6.5 h after the application of cytokinin. Only a very low level of in vivo hydroxylation can be demonstrated in half-seedlings treated for 6 h either with or without cytokinin but it begins to increase shortly after this time. A large increase in this activity by 16 h (independent of cytokinin) can be almost completely (79%) prevented by chloramphenicol (300 μM) although the drug increases accumulation of betacyanin. At this concentration about 30% of the protein synthesis in inhibited. In vitro tyrosine hydroxylation is, however, not reduced in half-seedlings treated with chloramphenicol during 16 h induction nor is extractable polyphenol oxidase reduced. It is concluded that chloramphenicol is inhibiting the synthesis of some protein essential for in vivo hydroxylation other than the activity measured during in vitro hydroxylation and that the inhibition of synthesis of 79% in vivo hydroxylation still leaves enough activity for maximum betacyanin synthesis.     

Inhibition of cytokinin action and of heat/aging induced potential for cytokinin action by inhibitors of membrane synthesis and function

Data to support the hypothesis that cytokinin action, in inducing the biosynthetic pathway involved in betacyanin synthesis in Amaranthus tri-color seedlings, is dependent on both membrane synthesis and function is presented. The experimental system included a pretreatment of heat shock (40°C) and aging of cotyledon explants. This produced the conditions necessary for the full expression of cytokinin potential during the subsequent betacyanin induction. Cerulenin, an inhibitor of fatty acid and sterol synthesis, inhibited both the heat-induced potential for cytokinin action and the benzyladenine-dependent induction itself. This was also true of metyrapone, an inhibitor of hydroxylation reactions involving cytochrome P450. Gammexane, an inhibitor of phospholipid turnover, impaired the heat-induced process but not the benzyladenine-dependent betacyanin accumulation. This was also the case with 2-isopropyl-4-dimethylamino-5-methyl phenyl-1-piperidine carboxylate methyl chloride, an inhibitor of the cyclization steps in sterol and gibberellin synthesis. Filipin at 100 micrograms per milliliter inhibited both processes, particularly the heat-induced potential. The effect of various steroids and fatty acids on induction is recorded together with experiments aimed at using them to reverse some of the inhibitions. The effect of cerulenin on heat-induced potential was partially reversed by preparations of Amaranthus lipids. Some reversal of the filipin effects was obtained with β-sitosterol and stigmasterol. It is concluded that menbrane synthesis is stimulated during the heat/aging pretreatment and during the induction and that some membrane function(s) is necessary for subsequent cytokinin action.     

Temperature-sensitive Responses of Red Light-dependent Betacyanin Synthesis

Amaranthus tricolor half-seedlings show a greater accumulation of betacyanin in response to a defined period of red light if given a pretreatment at an elevated temperature (40 C). Red light given before the shift to 40 C is ineffective. The maximum response is achieved after a 2-hour shift to 40 C and if the red light is given 1 hour after return to the germination temperature (25 C). The effect on red light induction of betacyanin synthesis and on fusicoccin induction by these conditions is similar, whereas the increase in cytokinin-dependent synthesis is greater. Both phytochrome-cytokinin synergism and fusicoccin-cytokinin synergism are changed in the same manner by this treatment. Phytochrome and fusicoccin responses are inhibited similarly by carbonyl-cyanide-p-trifluoromethoxy-phenylhydrazone.     

Replacement of light by dibutyryl-CAMP and CAMP in betacyanin synthesis

The effect of adenosine 3′,5′-cyclic monophosphate and its N⁶,O^2′- dibutyryl derivative (Bu₂-CAMP) on betacyanin formation in etiolated Amaranthus paniculatus seedlings was investigated. Both substances can replace the action of light in the synthesis of these pigments, the formation of which is controlled by phytochrome. The specificity of this mimicry is underlined by the observations that sodium butyrate does not promote any betacyanin formation and that theophylline enhances the effect of Bu₂-AMP. Puromycin inhibits the induction of betacyanin synthesis by Bu₂-CAMP just as it does the light-induced pigment formation. These findings suggest that phytochrome exerts its controlling role in the synthesis of betacyanins through the agency of CAMP.     

Reversal of abscisic acid-inhibited betacyanin synthesis by phenolic compounds in Amaranthus caudatus seedlings

Abscisic acid inhibits light-induced betacyanin synthesis in Amaranthus caudatus seedlings. Kinetin antagonizes ABA action and restores betacyanin synthesis. Similarly, phenolic compounds also antagonize ABA action and restore the synthesis of betacyanin. When present together phenolic compounds have an additive effect with kinetin in restoring ABA-inhibited betacyanin synthesis.     

Gibberellic acid and indole acetic acid compete in ethylene promoted abscission

Summary Gibberellic acid inhibition of betacyanin biosynthesis has been studied in Amaranthus caudatus L. using the pigment precursors L-tyrosine and L-dihydroxyphenylalanine. Precursors fed to gibberellic acid (GA) treated seedlings completely recovered betacyanin synthesis while the GA induced growth enhancement remained unaltered. Inhibition of betacyanin biosynthesis by GA is related to depletion of metabolites/amino acids and their diversion to support changed pattern of metabolism leading to growth.Abbreviations GA gibberellic acid - L-DOPA L-dihydroxyphenylalanine - Pr phytochrome red absorbing form - Pfr phytochrome, far-red absorbing form     

Effect of continuous far red on betaxanthin and betacyanin synthesis

Seedlings of Celosia plumosa under prolonged irradiation with far red light synthesize chlorophyll α and betaxanthin. Levulinic acid and 2,4-dinitrophenol, inhibitors of chlorophyll synthesis and cyclic photophosphorylation respectively, reduce betaxanthin synthesis. Pigment formation is also inhibited by actinomycin-D and puromycin, but is unaffected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of noncyclic photophosphorylation. These findings are evidence of the involvement of photosynthesis through cyclic photophosphorylation, in the far red HER associated with betaxanthin synthesis. Under continuous far red seedlings of Amaranthus tricolor synthesize only chlorophyll α. Lack of betacyanin formation is ascribed to the inactive status of the genes involved in the pigment synthesis.     

Anticytokinin activity of 4-substituted triazolo[4,5-d]pyrimidines and 4-substituted pyrazolo[3,4-d]pyrimidines

The synthesis and physiological activity of some novel 4-substituted triazolo[4,5-d]pyrimidines and 4-substituted pyrazolo[3,4-d]pyrimidines are described. Most of the compounds possessed high anticytokinin activity towards purine (benzyladenine) and phenylurea (4-PU-30) type cytokinins. 1-Benzyl-4-ethoxycarbonylpiperazinyl-1H-1,2,3-triazolo[4,5-d]pyrimidine almost completely removed cytokinin stimulated effects—betacyanin synthesis in Amaranthus caudatus cotyledons; growth of radish cotyledons and retention of chlorophyll in leaf explants. Some chemical structurephysiological activity relationships have been established.     

Betacyanin biosynthesis in the isolated hypocotyls bei Acetabularia mediterranea

Summary Isolated hypocotyls synthesize betacyanin after light exposure in Amaranthus caudatus L. Pigment synthesizing capacity is reduced in the hypocotyls with increased incubation of seedlings in dark after 24h. External feeding of precursors of betacyanin L-tyrosine and DOPA enhances pigment synthesis in the isolated hypocotyls to equal that of intact hypocotyls. Cotyledons are probably the source of precursors while both cotyledons and hypocotyls are the sites of betacyanin synthesis. Betacyanin synthesizing capacity is progressively lost from the base of the hypocotyl and precursors could not induce pigment synthesis in these regions.     

The effect of Benlate and cytokinins on the content of tobacco mosaic virus in tomato leaf disks and cucumber mosaic virus in cucumber cotyledon disks and seedlings

Tomato leaf disks were inoculated with tobacco mosaic virus (TMV) and floated for 7 days on solutions of kinetin and benzyladenine in the range 20-0-002 mg/1. Virus content was reduced at the higher and increased at the lower concentrations. Benlate and benomyl showed a peak of cytokinin activity in the Amaranthus betacyanin bioassay equivalent to c. 0–002 fig/l kinetin. At concentrations above 25 and 100 mg a.i./l for Benlate and benomyl respectively, both compounds increased the TMV content of tomato leaf disks. Cucumber mosaic virus (CMV) content in cucumber cotyledon disks was increased by Benlate and benomyl treatment (50–100 mg/1). Applied as a soil drench (50–500 mg a.i./l) when the plants were inoculated, Benlate increased the CMV content of infected seedlings. The number of starch-iodide lesions (a measure of susceptibility) was unaltered in cotyledons treated with Benlate 7 days before or immediately after inoculation. Infectivity of crude infective cucumber sap was unaffected by benomyl incorporation, whereas Benlate reduced infectivity at higher concentrations (1000–5000 mg/1). Under the experimental conditions described, Benlate, benomyl, benzyladenine and kinetin had no effect on the chlorophyll content of tomato leaf disks, and intact seedlings.     

Similarity between Cytokinin and Blue Light Inhibition of Cucumber Hypocotyl Elongation

The cytokinin benzyladenine inhibited endogenous hypocotyl elongation in intact etiolated seedlings of cucumber (Cucumis sativus L.). In hypocotyl segments, the inhibitory effect of benzyladenine on growth was clearly detectable in the presence of indoleacetic acid. Fusicoccin-induced elongation was unaffected by the presence of cytokinin. The effect of cytokinin on elongation of the segments was determined by measuring changes in fresh weight, a linear function of extension growth. The effect of benzyladenine on hypocotyl growth was at least as large in segments prepared from red-light-grown seedlings as in those from seedlings grown in total darkness. A comparison was made between the inhibitory effects of cytokinin and blue light. The use of the calcium chelator ethyleneglycol-bis(β-aminoethyl ether)-N, N′-tetraacetic acid indicated that calcium ions are required for manifestation of benzyladenine-induced inhibition.     

Nitrate reductase in agrostemma githago comparison of the inductive effects of nitrate and cytokinin

The effect of nitrate and cytokinin on the induction of nitrate reductase (NADH-nitrate oxidoreductase, EC 1.6.6.1) in embryos of Agrostemma githago was compared. Increased enzyme levels in response to treatment with the cytokinin benzyladenine were not correlated with a general stimulation of protein synthesis or a general reduction of protein breakdown. Actinomycin D did not inhibit the formation of nitrate reductase in response to nitrate or the cytokinin. Cycloheximide and puromycin inhibited the induction by the hormone to the same extent as, or even more than, the incorporation of [¹⁴C]leucine into protein. Induction of nitrate reductase by nitrate was much less susceptible to inhibition by cycloheximide and puromycin than induction of the enzyme by benzyladenine. When induction of nitrate reductase was carried out in the presence of ²H₂O, isopycnic equilibrium centrifugation in CsCl showed that incorporation of ²H into the enzyme had occured. The increase in the buoyant density of nitrate reductase was the same whether the enzyme was induced by nitrate or by benzyladenine, indicating that at least part of the nitrate reductase molecule was newly synthesized in both instances.     

A modifiedAmaranthus betacyanin test for cytokinin bioassay

A modified procedure for the extraction of betacyanin fromAmaranthus seedlings is described. Application of this modification increased the absorbance of cytokinin-treatedAmaranthus explants in most cases. The modifiedAmaranthus test is compared with the soybean callus test in the bioassay of kinetin, 6-benzylaminopurine, and 6(δ,δ-dimethylallyl) aminopurine.     

Studies of Rapidly Induced Wound Ethylene Synthesis by Excised Sections of Etiolated Pisum sativum L., cv. Alaska: IV. Requirement of a Water-soluble, Heat-stable Factor

The rate of wound ethylene synthesis was reduced by more than 85% when 9-millimeter subapical sections of etiolated 7-day-old Pisum sativum L., cv. Alaska seedlings were incubated in water during the 26-minute induction period prior to wound ethylene synthesis, but the rate of synthesis was unaffected if sections were incubated in water during the actual synthesis of wound ethylene. The characteristic timing of the wound response was unaffected by either treatment. The ability of various chemical solutions and aqueous plant extracts to alter the rate of wound ethylene synthesis was studied by first incubating subapical pea stem sections in solutions under anaerobic conditions (anaerobiosis delays the induction and synthesis of wound ethylene; Plant Physiol 61: 675-679), and then measuring wound ethylene synthesis after the tissue was transferred to air. Solutions of several reported precursors of ethylene synthesis, such as methionine, homoserine, or propanal, did not reverse the water-caused reduction of wound ethylene synthesis. A water-soluble, heat-stable factor in extracts from pea seedlings, and solutions of 23 nanomolar triacontanol, 10 micromolar kinetin, or 10 micromolar benzyladenine prevented the reduction of wound ethylene synthesis, but were ineffective if administered after an initial 15-minute anaerobic water incubation. This suggested that the active solutions may have only prevented the loss of some ephemeral, though necessary factor, rather than actually containing the substrate or inducer of wound ethylene synthesis. Attempts to isolate and characterize the active fraction from aqueous tissue extracts were unsuccessful. Free radical quenchers, inhibitors of protein synthesis, and rhizobitoxine, an inhibitor of ethylene synthesis from methionine, all reduced wound ethylene synthesis when administered in solutions which previously had maintained wound ethylene synthesis.     

Ca^2＋-Calmodulin is Involved in Betacyanin Accumulation Induced by Dark in C3 Halophyte Suaeda salsa

The C3 halophyte Suaeda salsa was used to investigate the roles of Ca^2＋, Ca^2＋ channels, and calmodulin （CAM） in betacyanin metabolism. Seeds of S. salsa were cultured in both the dark and light for 3 days. The fresh weight and betacyanin content were much higher in S. salsa seedlings formed in the dark than in seedlings formed in the light. The addition of Ca^2＋ to the half-strength MS nutrient solution promoted betacyanin accumulation in the dark, whereas Ca^2＋ depletion by EGTA suppressed the dark-induced betacyanin accumulation in shoots of S. salsa. The Ca^2＋ channel blocker LaCl3 also inhibited dark-induced betacyanin accumulation. The highest activity of CaM and the maximum betacyanin content decreased by 51% and 45%, respectively, in shoots of S. salsa seedlings treated with the potent CaM antagonist chlorpromazine in the dark. Furthermore, the other CaM antagonist N-（6-aminohexyl）-5-chloro-l-naphthalenesulfonamide （W-7） also inhibited the activity of CaM and dark-dependent betacyanin accumulation, whereas its less active structural analog N-（6-aminohexyl）- 1-naphthalenesulfonamide （W-5） had little effect on the responses to dark of S. salsa seedlings. These results suggest that Ca^2＋, Ca^2＋-regulated ion channels, and CaM play an important role in dark-induced betacyanin accumulation in the shoots of the C3 halophyte S. salsa.     

Effect of riboflavie and cyanide on blue light induction of betacyanin formation in Amamnthus caudatus seedlings

The role of phyto chrome and flavins in blue light induction of betacyanin formation was studied in etiolated, three-day-old Amaranthus caudatus L. seedlings, using the criterion of far-red reversibility and exogenously applied riboflavin and KCN. The effect of riboflavin was studied using high fluence rate blue light (42.7 :nmol m⁻²s⁻¹nm⁻¹ at 450 nm). When present in the incubation medium during illumination, riboflavin promoted the far-red reversibility with short light treatments and suppressed the inductive action of continuous illumiaation. If added after light treatments, it promoted betacyanin formation. The filtration of blue light through the riboflavin solution caused profound changes in light quality without affecting the far-red reversibility after 30 mm illumination. The effect of 1 mM KCN was tested with 70'% lower fluence of blue light. Cyanide caused the suppression of the inductive effect with 5 min blue light, which was accompanied by an enhancement of betacyanin induction by the terminal far-red light pulse. With 30 min blue light, however, it caused the appearance of far-red reversibility. The inductive effect of continuous blue illumination was slightly promoted by this Inhibitor. These results demonstrate that the effect of blue light on the pbyto chrome system is complex, whereas the physiological (inductive) action of the flavin triplet state is limited to low fluence, short blue light treatments.     

Ionic Regulation for Cytokinin-dependent Betacyanin Synthesis in Amaranthus Seedlings

Potassium ions at low concentrations stimulate cytokinin-dependent betacyanin synthesis in Amaranthus tricolor seedlings more than other alkali metal ions when tested as the chloride salts. The sequence of relative stimulation is K⁺ > Rb⁺ > (Na⁺ = Li⁺). Calcium and Mg²⁺ ions are inhibitory at concentrations > 1 millimolar when tested as chlorides. Anions also have an effect on the degree of alkali metal stimulation in the order PO₄³⁻ > NO₃⁻ > Cl⁻. The high activity of phosphate may be partly due to its chelating effect on inhibitory Ca²⁺ ions, or to effects on K⁺ uptake. A mixture of Na⁺ and K⁺ in the presence of phosphate is more effective than either cation alone. This result may be due either to effects on tyrosine transport or on the potassium uptake system. Phytochrome-dependent betacyanin synthesis shows the same stimulation by Na⁺ plus K⁺. The effect of a number of inhibitors of transport systems on betacyanin accumulation is reported. The possible role of the ionic environment of cells in their metabolic regulation is discussed, particularly in relation to cytokinin action.     

Activity and accumulation of cell division-promoting phenolics in tobacco tissue cultures

Dehydrodiconiferyl alcohol glucosides (DCGs) are derivatives of the phenylpropanoid pathway that have been isolated from Catharansus roseus L. (Vinca rosea) crown gall tumors. Fractions containing purified DCGs have been shown previously to promote the growth of cytokinin-requiring tissues of tobacco in the absence of exogenous cytokinins. In this study, we utilized synthetic DCG isomers to confirm the cell division-promoting activity of DCG isomers A and B and show that they neither promote shoot meristem initiation on Nicotiana tabacum L., cv Havana 425, leaf explants nor induce betacyanin synthesis in amaranth seedlings. Analysis of cultured tobacco pith tissue demonstrated that DCG accumulation was stimulated by cytokinin treatment and correlated with cytokinin-induced cell division. Thus, the accumulation of metabolites that could replace cytokinin in cell division bioassays is stimulated by cytokinins. These data support the model that DCGs are a component of a cytokinin-mediated regulatory circuit controlling cell division.