The effects of hormones and inhibitors on amaranthin synthesis in seedlings of Amaranthus tricolor

Summary Both gibberellic acid and abscisic acid inhibit the light induced synthesis of amaranthin in Amaranthus tricolor seedlings. The auxin, indolyl-3-acetic acid has no effect. The protein/RNA inhibitors, cycloheximide and 8-azaguanine, also reduced the levels of amaranthin produced.     

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.     

Effects of light and kinetin on amaranthin synthesis induced by cAMP

The effects of cyclic 3′,5′-adenosine monophosphate (cAMP) on amaranthin synthesis in the dark, or in the presence of kinetin or light were investigated in isolated cotyledons of Amaranthus tricolor and A. caudatus. The results suggest that sites or modes of action of cAMP and kinetin are not separated and differ from those of light and that the nucleotide cannot be considered a messenger involved in amaranthin formation stimulated by kinetin or by light.     

Temperature-dependent Expression of Betacyanin Synthesis in Amaranthus Seedlings

Two phenomena related to temperature effects have been observed during the induction of betacyanin synthesis by a cytokinin (benzyladenine) in Amaranthus tricolor seedlings. One is a total inhibition of betacyanin accumulation at a temperature (39 C) at which seedling growth is unimpaired, and where there is still adequate uptake of benzyladenine. The other is the apparent induction of a higher potential for subsequent betacyanin synthesis following pretreatment of the seedlings at an elevated temperature.     

Effect of a cytokinin antagonist on cytokinin and light-dependent amaranthin synthesis inAmaranthus Tricolor seedlings

InAmaranthus tricolor seedlings, amaranthin synthesis can be induced under the effect either of a cytokinin or of white light. The 3-methyl-7-(n-pentylamino)pyrazolo(4,3-d)pyrimidine (PAMPP), a cytokinin analog that strongly inhibits the growth of tobacco callus, antagonizes the stimulating effect of cytokinin as well as stimulation by light. In dose-response terms, the inhibitory effect of PAMPP was described as competitive with respect to N⁶-benzyladenine (b⁶Ade) or light. The inhibition by PAMPP of the b⁶Ade amaranthin test response or the inhibition by this cytokinin analog of the light amaranthin test response were both reversed by either subsequent light or b⁶Ade treatment.     

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.     

Effect of a cytokinin antagonist on cytokinin and light-dependent amaranthin synthesis inAmaranthus Tricolor seedlings

InAmaranthus tricolor seedlings, amaranthin synthesis can be induced under the effect either of a cytokinin or of white light. The 3-methyl-7-(n-pentylamino)pyrazolo(4,3-d)pyrimidine (PAMPP), a cytokinin analog that strongly inhibits the growth of tobacco callus, antagonizes the stimulating effect of cytokinin as well as stimulation by light. In dose-response terms, the inhibitory effect of PAMPP was described as competitive with respect to N⁶-benzyladenine (b⁶Ade) or light. The inhibition by PAMPP of the b⁶Ade amaranthin test response or the inhibition by this cytokinin analog of the light amaranthin test response were both reversed by either subsequent light or b⁶Ade treatment.     

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.     

Interaction of kinetin and calcium in relation to their effect on stimulation of ethylene production

Application of kinetin and Ca²⁺ caused a striking synergistic increase in ethylene production by mung bean (Phaseolus aureus Roxb) hypocotyl segments. The effect of kinetin on Ca²⁺ uptake and of Ca²⁺ on the uptake and metabolism of kinetin in relation to their effect on ethylene production was studied. Tracer experiments showed that kinetin greatly increased the uptake of ⁴⁵Ca²⁺ after 6 hours of incubation. Reciprocally, Ca²⁺ stimulated the uptake of kinetin-8-¹⁴C and remarkably enhanced the metabolism of kinetin-8-¹⁴C into several polar metabolites. Consequently, the quantity of free kinetin-8-¹⁴C remaining in Ca²⁺-treated segments was much less than in control segments. A possible mechanism accounting for the synergism between kinetin and calcium on ethylene production is discussed.     

Betacyanin decolourizing enzyme in Amaranthus tricolor seedlings

The characterization and partial purification of an enzyme from Amaranthus tricolor which decolourizes betacyanin are described. The enzyme occurs in greatest amounts in the roots and in 3.5-4-day-old seedings. Preparation from an acetone powder of roots results in a more active and more stable enzyme than that obtained from crude buffer extraction. The activity is in the 130 000 g supernatant from sucrose-buffer extraction. It has a pH optimum of 3.4 K_m towards amaranthin of 3.1 × 10^?6 M and towards betanin of 3.5 × 10^?6 M, and is inhibited by lack of oxygen, and by azide, diethyldithiocarbamate, thiourea, dithiothreitol and cysteine. The product of the reaction has the spectral and electrophoretic properties of betalamic acid. The possibility of enzymic decolourization of betacyanin during acetic acid extraction used for assay of the pigment in the Amaranthus bioassay for cytokinins needs to be recognized.     

Dimethyl sulfoxide: A solvent for cytokinins in theAmaranthus bioassay

Dimethyl sulfoxide present in the agar medium at concentration 0.2 % (v/v) and lower does not inhibit cytokinin-induced betacyanin synthesis in theAmaranthus caudatus seedlings. The activity of kinetin, N⁶-(Δ²-isopentenyl)adenine andtrans- zeatin is the same when these cytokinins are dissolved in either water or dimethyl sulfoxide and incorporated into the medium after autoclaving. A simple method is described which allows the cytokinin activity of slightly water-soluble and thermolabile compounds,e.g. aromatic urea and thiourea derivatives, to be determined in theAmaranthus bioassay.     

Interaction of Amaranthin with the Electron Transport Chain of Chloroplasts

The electron paramagnetic resonance method was used to study the interactions of amaranthin with isolated class B chloroplasts from broad bean (Vicia faba L.) and amaranth (Amaranthus tricolor L.) during the light-driven electron and proton transport. Amaranthin was shown to interact with electron transport chain of chloroplasts at the PS II level; it also affects the electron transport near PS I. At the same time, amaranthin had no significant inhibitory effect on the light-dependent formation of the transmembrane pH gradient.     

Calmodulin-binding drugs affect responses to cytokinin, auxin, and gibberellic Acid

Trifluoperazine, a phenothiazine tranquilizer, and tetracaine, a local anesthetic, have been found to inhibit a variety of plant hormone responses at concentrations compatible with their known inhibition of Ca²⁺-calmod-ulin-dependent enzyme activities. Among these responses are cytokinin-dependent betacyanin synthesis and increase in fresh weight in Amaranthus tricolor cotyledons, auxin-dependent increase in length of wheat coleoptile segments and gibberellic acid-dependent induction of α-amylase synthesis in barley aleurone layers. The reversibility of some of these inhibitory effects has been demonstrated, indicating that, up to a point, a generalized membrane destruction can be ruled out. The evidence, taken in conjunction with numerous examples from the literature showing calcium involvement in the action of all of the plant hormones, support a unifying theory of hormone action.     

Accumulation of Cytokinin-Induced Betacyanin in Specific Cells of Amaranthus tricolor Seedlings

The accumulation of betacyanin, in dark-grown Amaranthus tricolorseedlings, in response to cytokinins or red light, occurs mainlyin two specific tissues, the lower epidermal cells of the cotyledons(with the exception of guard cells), and the endodermis of thehypocotyl. The possible significance of this ‘spatialpattern of competence’ is discussed, together with theconcept of target cells in relation to plant hormones. The effect of removing exogenously supplied cytokinin at varioustimes during a 24 h induction period is reported. There is noevidence that cytokinins act by a ‘triggering’ effectwith a long half life, the response in the target cells beingthe same as that expected from the amount of cytokinin and cytokininmetabolite remaining in the tissue at the time of extraction.Either continuous presence of cytokinin is needed or any triggeraction is short lived, and continuous ‘re-triggering’is needed to achieve maximum response. Key words: Amaranthus tricolor, Betacyanin synthesis, Cytokinin action, Target cells     

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 hormonal control of betacyanin synthesis in Amaranthus caudatus

Gibberellic acid (GA₃) inhibits amaranthin synthesis whereas the growth retardant, phosphon D, enhances pigment levels in A. caudatus seedlings exposed to light. No effect was observed on chlorophyll and carotenoid synthesis. Radioactive tyrosine and DOPA were incorporated into amaranthin. The specific activity of amaranthin synthesised in the presence of ¹⁴C-tyrosine or ¹⁴C-DOPA in seedlings treated with GA₃ is higher than water controls. The specific activity of pigment from phosphon D treated tissue is relatively low. GA₃ treated tissue has lower active tyrosine and DOPA pools compared to phosphon treated seedlings. Tyrosine and DOPA-oxidase activity increases in GA₃ treated and H₂O control seedlings exposed to light. Kinetin stimulates the synthesis of amaranthin in dark-grown seedlings and this is not overcome by simultaneous GA₃ application. Dark-grown seedlings treated with different kinetin concentrations and incubated in ¹⁴C-tyrosine synthesise radioactive amaranthin of similar specific activity. Kinetin treatment of dark-grown seedlings brings about an increased tyrosine and DOPA-oxidase activity. The results indicate that GA₃ controls the production and/or availability of tyrosine whereas kinetin can mimic light treatment and controls the utilisation of tyrosine probably by bringing about the synthesis or activation of tyrosine and DOPA-oxidase protein.     

The Dual Effects of Ca2+ on Binding of the Glycolytic Enzymes,Phosphofructokinase and Aldolase,to Muscle Cytoskeleton

We show here that in rat diaphragm muscle, a short time of incubation with the Ca²⁺-ionophore A23187 induced an increase in cytoskeleton-bound phosphofructokinase (EC 2.7.1.11) and aldolase (EC 4.1.2.13), whereas a longer period of incubation, which causes a pathological rise in intracellular Ca²⁺, induced a decrease in bound enzymes. Lactate concentration correlated with both phases of Ca²⁺ action on the binding of the enzymes. The increase in cytoskeleton-bound enzymes could be prevented by treatment with the calmodulin antagonists trifluoperazine or CGS 9343B (a novel, potent, and selective inhibitor of calmodulin activity). These results suggest that calmodulin is involved in the Ca²⁺-induced binding of the enzymes to muscle cytoskeleton.     

The influence of light on the cytokinin content ofAmaranthus seedlings

The influence of light, particularly blue and red light, on the cytokinin content of seedlings ofAmaranthus caudatus was studied. Cytokinin content was determined by two different bio-assays (amaranthin accumulation byAmaranthus seedlings and mtrate-reductase activity ofAgrostemma embryos). In both bio-assays similar results were obtained. Oytokinin content is increased, especially by blue light. It is suggested that especially blue light promotes amaranthin accumulation inAmaranthus seedlingsvia the increase of cytokinin content of tissues. The results support our hypothesis on cytokinin action.     

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.     

Genetic study of the role of calcium ions in the cell division cycle of Saccharomyces cerevisiae: a calcium-dependent mutant and its trifluoperazine-dependent pseudorevertants

Summary A cal1-1 mutant of the yeast Saccharomyces cerevisiae showing Ca²⁺-dependent growth was isolated. Its growth continued exponentially in Ca²⁺-rich medium, but stopped in Ca²⁺-poor medium at 37°C. Mg²⁺ ions could not replace Ca²⁺ ions. In Ca²⁺-poor medium, the mutant cells stopped growing homogeneously at the stage of cell division cycle with a tiny bud. The nucleus in these arrested cells was in the G2 stage, judging from observation after nuclear staining and determination of the DNA content. Trifluoperazine-dependent pseudorevertants, which could grow in the presence of 20 M to 80 M trifluoperazine in Ca²⁺-poor medium at 37°C, were obtained from this cal1-1 mutant. The suppressor mutation, tfrl, itself conferred trifluoperazine resistance. Other calmodulin inhibitors structurally unrelated to trifluoperazine had similar effects to trifluoperazine on these pseudorevertants. These results suggest that Ca²⁺ ions and a calmodulin play important roles in the yeast cell division cycle at the stage of bud growth and nuclear division.Abbreviations Tfp trifluoperazine - DAP1 46-diamidino-2-phenylindole - EMS ethyl methanesulfonate - PD parental ditype - NPD nonparental ditype - T tetratype