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.     

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.     

Interactions of Abscisic Acid, Cytokinin and Gibberellin in the Control of Betacyanin Synthesis in Seedlings of Amaranthus caudatus

In de-rooted seedlings of Amaranthus caudatus L., betacyanin synthesis induced by white light or cytokinin was inhibited by abscisic acid (ABA) or a mixture of gibberellins A₄ and A₇ (GA_4/7). The GA_4/7 and ABA effects were additive. Thus ABA inhibited the cytokinin action but had no effect on the gibberellin response.     

Regulatory mechanisms of biosynthesis of betacyanin and anthocyanin in relation to cell division activity in suspension cultures

Regulatory mechanisms of betacyanin biosynthesis in suspension cultures of Phytolacca americana and anthocyanin in Vitis sp. were investigated in relation to cell division activity.Betacyanin biosynthesis in Phytolacca cells clearly shows a positive correlation with cell division, as the peak of betacyanin accumulation was observed at the log phase of batch cultures. Incorporation of radioactivity from labelled tyrosine into betacyanin also showed a peak at early log phase. Aphidicolin, an inhibitor of DNA synthesis, and propyzamide, an antimicrotubule drug, reduced betacyanin accumulation and inhibited the incorporation of radioactivity from labelled tyrosine into betacyanin at concentrations which were inhibitory to cell division. Both inhibitors reduced the incorporation of radioactivity from labelled tyrosine to 3,4-dihydroxyphenylalanine (DOPA), but the incorporation of labelled DOPA into betacyanin was not affected. These results suggest that the conversion of tyrosine to DOPA is coupled with cell division activity.In contrast, the anthocyanin accumulation in Vitis cells showed a negative correlation with cell division. Accumulation occurred at the stationary phase in batch cultures when cell division ceased. Aphidicolin or reduced phosphate concentration induced a substantial increase in anthocyanin accumulation as well as the inhibition of cell division. Chalcone synthase (CHS) activity increased at the time of anthocyanin accumulation. Northern blotting analysis indicated that changes in CHS mRNA levels corresponded to similar changes in enzymatic activity. The pool size of endogenous phenylalanine was low during active cell division, but increased before anthocyanin began to accumulate and concomitantly with increasing levels of CHS mRNA. Exogenous supply of phenylalanine at the time of low endogenous levels induced the elevation of CHS mRNA and anthocyanin accumulation. These results indicate that the elevation of endogenous phenylalanine levels, when cell division ceases, may cause the increase in CHS mRNA levels, resulting in increased CHS activity and subsequently in anthocyanin accumulation in Vitis suspension cultures.Abbreviations CHS chalcone synthase - CHFI chalcone flavanone isomerase - DOPA 3,4-dihydroxyphenylalanine - PAL phenylalanine ammonia lyase     

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.     

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.     

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.     

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.     

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.     

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     

Rapid activation of specific phospholipase(s) D by cytokinin in Amaranthus assay system

The suggested link between intracellular cytokinin signaling and phospholipase D (PLD, EC 3.1.4.4.) activity (Romanov et al. 2000, 2002) was investigated. The activity of PLD in the early period of cytokinin action was studied in vivo in derooted Amaranthus caudatus seedlings, using the level of phosphatidylbutanol production as a measure of PLD activity. Rapid activation of phosphatidylbutanol synthesis was demonstrated as early as within 5 min of cytokinin administration. Neomycin, a known phosphatidylinositol‐4,5‐bisphosphate (PIP₂) antagonist, strongly repressed both physiological cytokinin effect and cytokinin‐dependent PLD activation. N‐acylethanolamine (NAE 12), an inhibitor of α‐class PLD, did not influence significantly cytokinin effect on Amaranthus seedlings. Together, results suggest the involvement of PIP₂‐dependent non‐class α‐PLD in the molecular mechanism of cytokinin action.     

Regulation of cytokinin oxidase activity in tobacco callus expressing the T-DNA ipt gene

There are indications that the cytokinin content in transgenic tissues expressing the cytokinin biosynthetic ipt gene is under metabolic control, which prevents the accumulation of cytokinins to lethal levels. The objective of this study was to investigate the relationships between the content of endogenous cytokinins and the activity of cytokinin oxidase (which is believed to be a copper-containing amine oxidase, EC 1.4.3.6.) in ipt transgenic tobacco callus. In addition, the effect of exogenously applied N-benzyladenine (BA) on this relationship was examined. Endogenous cytokinin concentrations were measured in callus of Nicotiana tabacum L. cv. Petit Havana SRI transformed with the ipt of Agrobacterium tumefaciens under the control of a light-inducible promoter and in non-transformed tissue using LC-tandem mass spectrometry. The activity of cytokinin oxidase was estimated by measuring the conversion of [2,8-³H]N⁶-(Δ²-isopentenyl)adenine to [³H]adenine by enzyme preparations in vitro. The 14-day-old ipt-transformed callus contained a 25-fold higher amount of cytokinins as compared to the non-transformed tissue. Mainly zeatin- and dihydrozeatin-types of cytokinins (free bases, ribosides, nucleotides and O-glucosides) accumulated in the ipt transgenic tissue. The cytokinin pool of both ipt-transformed and non-transformed tissues consisted predominantly of cytokinins that are either resistant to cytokinin oxidase attack (nucleotides and O-glucosides of cytokinins and cytokinins bearing N⁶-saturated side chain) or have a low affinity for the enzyme (zeatin and its riboside). The former represented 71.6 and 74.8% and the latter 27.7 and 24.4% of the pool of endogenous cytokinins in ipt-transformed and non-transformed tissues, respectively. Enzyme preparations from ipt-transformed tissue exhibited 1.5-fold higher cytokinin oxidase activity compared with that observed in control tissues. Application of exogenous BA affected the total levels of cytokinins of the two tissue lines in different ways. The cytokinin content increased by 1.7- and 1.5-fold in ipt-transformed tissues 6 and 12 h after BA application, respectively, while it declined in the non-transformed control by 1.6- to 2.0-fold between 3 and 12 h after BA application. The increase in cytokinin content in the ipt callus is due to an increase of zeatin- and dihydrozeatin-type cytokinins (nucleotides, ribosides and free bases) leading to an enhanced accumulation of O-glucosides after 12 h. Following BA treatment, the cytokinin oxidase activity increased up to 1.8-fold in ipt-transformed and 1.6-fold in non-transformed tissues. The levels of isopentenyl-type cytokinins were near the detection limit; however, the enhancement of cytokinin oxidase activity after BA treatment in both tissue lines was correlated with the content of preferred substrate of the enzyme, N⁶-(Δ²-isopentenyl)adenosine.     

Cytokinin activities ofN 6-benzyladenosine derivatives hydroxylated on the side-chain phenyl ring

Cytokinin activities ofN ⁶-benzyladenosine (bzl⁶Ado) and its derivatives hydroxylated on the side chain phenyl ring inortho, meta, andpara positions were compared in four bioassays based on stimulation of growth of tobacco callus, retention of chlorophyll in excised wheat leaves, dark induction of betacyanin synthesis inAmaranthus cotyledons, and release of lateral buds of pea from apical dominance. In all these bioassays hydroxylation of the phenyl ring of bzl⁶Ado inortho andpara positions significantly decreased cytokinin activity. Compared with bzl⁶Ado, the activity was decreased about 10× in the tobacco callus bioassay and wheat leaf chlorophyll retention test, 100× in theAmaranthus betacyanin bioassay, and 20× and 200×, respectively, in the pea bud test. Hydroxylation of the phenyl ring inmeta position increased activity in the tobacco callus, and wheat leaf chlorophyll retention bioassays, 9× and 1.7×, respectively, decreased activity about 2.4× in the pea bud test and was without effect in theAmaranthus bioassay. Cytokinin activity of themeta hydroxy derivative,N ⁶-(m-hydroxybenzyl) adenosine, was as high as that oftrans-zeatin in all four bioassays. Possible regulation of biological activities of cytokinins by positionally specific hydroxylation of the side chain phenyl ring is discussed with respect to the reported occurrence of natural purinyl cytokinins with aromatic side chains.     

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.     

Effects of Short-term Red Radiation and Choline Compounds on Cytokinin Content, Chlorophyll Accumulation and Photomorphogenesis in Wheat Seedlings

Effects of choline compounds (2-chloroethyltrimethylammonium chloride and 2-ethyltrimethylammonium chloride) as well as red radiation (R) pulse on the dynamics of cytokinin changes, growth and chlorophyll (a + b) accumulation were studied during the growth and greening of etiolated wheat seedlings (Triticum aestivum L., var. Mironovskaya-808). The seedlings were grown for 120 h in the dark and then exposed for 72 h to white light. Pre-treatment of caryopses with cholines and pre-irradiation of etiolated seedlings with R inhibited elongation of both coleoptile and first leaf; but the same factors accelerated these growth responses when seedlings were exposed to white light. Chlorophyll (Chl) accumulation and the first leaf appearance from coleoptile were accelerated by the pre-treatments as well. Far-red radiation (FR) reversed all effects of R but choline pre-treatment eliminated partly R/FR photoreversibility. Two compounds with high cytokinin activity (tested on a fresh weight basis by the bioassay with Amaranthus caudatus L.) were found in shoots and first leaves. One of them had R_f, UV absorbance spectrum and the biological activity similar to N⁶-(Δ²-isopentenyl)adenosine. Another cytokinin-like substance was not identified with the used standards. Stimulation of greening by R pulse and cholines was accompanied with accelerated accumulation of both cytokinin-like substances. We conclude that the influence of R and cholines on the concentration of substances with cytokinin activities detected in the leaves might be involved in the stimulation of Chl accumulation.     

A modified Amaranthus betacyanin bioassay for the rapid determination of cytokinins in plant extracts

Summary Pure cytokinin standards and celery seed extracts containing cytokinin activity were bioassayed using a modified Amaranthus betacyanin bioassay. The assay is very rapid and requires no special sterile precautions.     

Effect of Hypocotyl Excision on Red Light-Mediated Betacyanin Formation in Amaranthus Seedlings

The effect of hypocotyl excision on red light-mediated betacyanininduction in cotyledons of Amaranthus caudatus L. was studiedusing etiolated, 3-day-old half-seedlings and isolated cotyledons.The removal of the hypocotyl promoted betacyanin formation undersafelight conditions in a manner competitive with brief (5 mins),but additive with prolonged (6 h) red illumination. If a papersupport was provided in order to improve the aeration, betacyaninformation in safelight conditions was further stimulated, reducingthe inductive effect of brief, but not changing the action ofprolonged red illumination. These results demonstrate that betacyaninphotoregulation is restricted to cotyledons of A. caudatus seedlings,with no evidence for transmission of light signals between differentorgans. Excision and aeration appear to promote selectivelya very low fluence response (VLFR) induced by safelight, suggestingdifferent mechanisms of phytochrome phototransduction underVLFR, low fluence response induced by brief saturating red light(LFR) and high irradiance reaction (HIR) occurring under prolongedred illumination. Amaranthus caudatus L, betacyanin photoregulation, red light, very low fluence phytochrome reaction     

Activities of enzymes of polyphenol metabolism inPhaseolus aureus seedlings germinated in the presence of 2-Chloroethylphosphonic acid

The plant growth regulator 2-ohloroethylphosphonic acid inhibited the elongation of growth inPhaseolus aureus seedlings. In comparison to the control, the polyphenol oxidase and peroxidase activity of treated seedlings was low up to 24 and 48 h of germination, respectively and that of phenylalanine ammonia-lyase and tyrosine ammonia-lyase was slightly less at 120 h and that of α- and β-glucosidases were less at 48 and 72 h, respectively. At other stages of germination, it greatly stimulated the activities of these enzymes. Part of Ph. D. dissertation submitted by Y. K. Arora to Punjab Agricultural University, Ludhiana, India     

Regulation of Cytokinin Oxidase Activity in Callus Tissues of Phaseolus vulgaris L. cv Great Northern

The regulation of cytokinin oxidase activity in callus tissues of Phaseolus vulgaris L. cv Great Northern has been examined using an assay based on the oxidation of N⁶-(Δ²-isopentenyl)adenine-8-¹⁴C (i⁶ Ade-8-¹⁴C) to adenine. Solutions of exogenous cytokinins applied directly to the surface of the callus tissues induced relatively rapid increases in cytokinin oxidase activity. The increase in activity was detectable after 1 hour and continued for about 8 hours, reaching values two- to three-fold higher than the controls. The cytokinin-induced increase in cytokinin oxidase activity was inhibited in tissues pretreated with cordycepin or cycloheximide, suggesting that RNA and protein synthesis may be required for the response. Rifampicin and chloramphenicol, at concentrations that inhibited the growth of Great Northern callus tissues, were ineffective in inhibiting the increase in activity. All cytokinin-active compounds tested, including both substrates and nonsubstrates of cytokinin oxidase, were effective in inducing elevated levels of the enzyme in Great Northern callus tissue. The cytokinin-active urea derivative, Thidiazuron, was as effective as any adenine derivative in inducing this response. The addition of Thidiazuron to the reaction volumes used to assay cytokinin oxidase activity resulted in a marked inhibition of the degradation of the labeled i⁶ Ade-8-¹⁴C substrate. On the basis of this result, it is possible that Thidiazuron may serve as a substrate for cytokinin oxidase, but other mechanisms of inhibition have not yet been excluded.     

Correlation of betacyanin synthesis with cell division in cell suspension cultures of Phytolacca americana

In suspension cultures of Phytolacca americana , betacyanin accumulation was reduced when cell division was inhibited by treatment with various inhibitors of DNA synthesis or anti-microtubule drugs. Aphidicolin (APC), an inhibitor of DNA synthesis, reduced the incorporation of radioactivity from labeled tyrosine into betacyanin, but the incorporation of radioactivity from labeled 3,4-dihydroxyphenylalanine (DOPA) into betacyanin was not affected by similar treatments. Propyzamide, another anti-microtubule drug, reduced incorporation of radioactivity from tyrosine and DOPA into betacyanin. However, the rate of incorporation from DOPA was higher than that from tyrosine. The results suggest that inhibition of betacyanin accumulation in Phytolacca americana cells by APC and propyzamide is due to suppression of the reaction converting tyrosine to DOPA, which may be closely related to cell division.