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.     

GA,ABA, phenol interaction in the control of growth: Phenolic compounds as effective modulators of GA-ABA interaction in radish seedlings

Abscisic acid, a potent growth inhibitor inhibits hypocotyl growth ofRaphanus sativus seedlings. Phenolic compounds,viz., trans-cinnamic acid, chlorogenic acid, ferulic acid, salicylic acid, tannic acid and quercetin when applied with ABA, antagonize ABA action and restore normal seedling growth. Gibberellic acid promotes hypocotyl growth and on combined application with ABA, the ratio of their concentrations determines the course of the resultant growth. This interaction can be modulated by phenolic compounds. Phenolic compounds in low concentrations when present together with GA and ABA, favour GA-induced growth by antagonizing the inhibitory influence of ABA. The inhibitory action of abscisic acid on a wide range of growth processes is so far known to be reversed only by growth promoting hormones,viz., IAA, GA and cytokinins. Antagonistic action of phenolic compounds towards ABA, and increasing the action of GA when present together with GA and ABA, establishes a dual role to this class of compounds; balancing the effect of both growth promoting and growth inhibiting hormones. Part I.     

Effect of abscisic acid on betacyanin leakage from plant tissues

Effect of abscisic acid on cell permeability in leaves ofIresine u allisi hort. and roots ofBeta vulgaris L. were examined. An increase of betacyanin leakage from leaf cells was shown by ABA at 10⁻⁴, 10⁻⁷ or 10⁻⁹ M concentrations in water solution at 25 °C. The efflux of batacyanin from tissues did not change during the joint action of ABA and PEG 1000. ABA could lower the betacyanin leakage fromIresine leaves and beet-root slices under severe osmotic stress, as was found by deplasmolysis. The results suggest that ABA elicits some alteration in density of tonoplast membranes under dehydration. Presented at the International Symposium “Plant Growth Regulators” held on June 18–22, 1984 at Liblice, Czechoslovakia.     

Abscisic acid in the plants-pathogen interaction

Information available concerning the role of ABA in the interaction between plants and pathogenic microorganisms allows a conclusion that this phytohormone is required for plant defense. For the development of plant resistance, short-term increases in the ABA level are of importance during the early stages of plant interaction with pathogens, which trigger anti-stress programs in plants, primarily related to the synthesis of callose. At the same time, high ABA concentrations maintained for a long time reduce efficiency of defense systems controlled by salicylic and jasmonic acids and ethylene. ABA was shown to suppress expression of some genes of defense proteins, including those involved in the synthesis and metabolism of phenolic compounds and lignin. ABA is evidently involved in plant defense mechanisms against pathogens as a regulatory element.     

Inhibition by phenolic compounds of cytokinin-stimulated betacyanin synthesis inAmaranthus caudatus

A number of phenolic compounds have been tested for ability to inhibit the cytokinin-induced synthesis of betacyanin inAmaranthus caudatus cotyledons. Under the conditions employed, the compounds responded thus: (1) no inhibition with up to 1 mg ml^-1 (quercetin 3-rhamnosylglucoside and chlorogenic acid), (2) partial or no inhibition up to 0.1 mg ml^-1 with greater inhibition at 1 mg ml^-1 (phloridzin, arbutin, caffeic acid, p-hydroxybenzoic acid and 3,4-dihydroxyphenylalanine) and (3) partial or no inhibition up to 0.1 mg ml^-1 with complete, inhibition at 1 mg ml^-1 (o-coumaric,m-coumaric,p-coumaric, protocatechuic and ferulic acids and phenylalanine). The results show that if theAmaranthus betacyanin bioassay for cytokinins is to give reliable results, then certain contaminating phenolics must be removed beforehand; some difficulties involved in this are briefly discussed.     

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.     

Water deficit and exogenous ABA significantly affect grape and wine phenolic composition under in field and in-vitro conditions

Phenolic compounds are components responsible of the sensorial characteristics of red wine. Indeed, they present important properties for human health. In the present work, the effects of combined in field treatments (water-stress and exogenous ABA) on phenolic accumulation were evaluated for berries and wine. The responses were assessed by UV–Vis and CZE. A differential phenolic regulation was observed under the same conditions and good correlations were achieved for grape and wine. ABA appears to regulate the content of each phenols under study depending on plant water-status. Although the effects of water-stress and exogenous ABA were similar in magnitude, our results support evidence suggesting that both affect different metabolic pathways. ABA supply increased catechin and malvidin synthesis for both water statuses while the resveratrol was enhanced only for water-stress. Indeed, ABA reduced the quercetin content for both water statuses. In vitro assays were carried out to estimate the effects of combined treatments (temperature-exogenous ABA) on anthocyanin accumulation in berries and pulp. These tests demonstrated that the effect of ABA was dependent on the hormonal level (207% for 0.5 g L⁻¹ and 307% for 1 g L⁻¹ of ABA). Effect of temperature varied according to the phenological state. In pulps, we detected an increase of anthocyanin after ABA treatments.     

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.     

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.     

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.     

Involvement of putative chemical wound signals in the induction of phenolic metabolism in wounded lettuce

Cutting leaves of Romaine lettuce ( Lactuca sativa L. cv. Longifolia) produces a wound signal that induces the synthesis of phenylalanine ammonia lyase (PAL, EC 4.3.1.5) and the accumulation of phenolic compounds in cells up to 2 cm from the site of injury, and tissue browning near the site of injury. The response of leaves within a head of Romaine lettuce to putative chemical wound signals [abscisic acid (ABA), jasmonate (JA) and methyl jasmonate (MeJA)] differed significantly with leaf age. Exposure of harvested heads of lettuce to ABA, JA, MeJA, or salicylic acid (SA) did not induce changes in PAL activity, the concentration of phenolic compounds or browning in mature leaf tissue that was similar to the level induced by wounding. Methyl jasmonate applied as vapour (10, 100 or 1000 µl kg⁻¹ FW), or as an aqueous spray or dip (0.01–100 µ M ) at 5 or 10°C did not produce an effect on PAL activity or browning that differed significantly from the untreated controls. In contrast, JA, MeJA and SA did induce elevated levels of PAL activity in younger leaves. However, the levels induced were far lower than those induced by wounding. Wound induced phenolic metabolism in mature leaves appears to be induced by different signals than those functioning in young leaves.     

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.     

Stimulation of betacyanin synthesis through exogenous methyl jasmonate and other elicitors in suspension-cultured cells of Portulaca

Betacyanin production in suspension-cultured cells of Portulaca was significantly enhanced by both abiotic and biotic elicitors. Betacyanin levels increased 1.3 and 1.5-fold over the controls in the presence of two abiotic elicitors (20 mumol/L CuSO4 and 100 mumol/L FeEDTA) and increased 1.8 and 1.6-fold in the presence of two biotic elicitors (0.5 mg/L beta-glucan and 0.5 mg/L chitosan). Maximum betacyanin synthesis with the two most effective elicitors was obtained when cultures were treated on day 1 and day 0 by beta-glucan and FeEDTA, respectively. A concentration-dependent response was exhibited by cultures treated with exogenous methyl jasmonate (MJ). MJ alone at 0.1 mumol/L caused a 2.6-fold increase in betacyanin synthesis when administered to the suspension culture on day 3. However, no additive effect on betacyanin accumulation was observed in treatments, which combined MJ and beta-glucan or FeEDTA. Treatment with ibuprofen (IB), an inhibitor of jasmonate biosynthesis, reduced the level of betacyanin in cells cultured in standard medium at all concentrations tested (25, 50, 100 mumol/L). The effect of IB on betacyanin synthesis in the cells treated with MJ or beta-glucan, however, differed with the IB concentration applied. The two higher concentrations (50 and 100 mumol/L) of IB significantly reduced the betacyanin content while the lower concentration (25 mumol/L) did not show an adverse effect on the betacyanin enhancement triggered by MJ or beta-glucan. Our findings suggest that, in suspension-cultured cells of Portulaca, an MJ-mediated signal transduction pathway prominently exists in betacyanin synthesis. This pathway seems to act antagonistically towards beta-glucan-mediated signaling. As far as we know this is the first report on the elevation of betacyanin level by jasmonate or other elicitors in cell suspension cultures.     

Abscisic acid is involved in phenolic compounds biosynthesis,mainly anthocyanins,in leaves of Aristotelia chilensis plants (Mol.) subjected to drought stress

Abscisic acid (ABA) regulates the physiological and biochemical mechanisms required to tolerate drought stress, which is considered as an important abiotic stress. It has been postulated that ABA might be involved in regulation of plant phenolic compounds biosynthesis, especially anthocyanins that accumulate in plants subjected to drought stress; however, the evidence for this postulate remains elusive. Therefore, we studied whether ABA is involved in phenolic compounds accumulation, especially anthocyanin biosynthesis, using drought stressed Aristotelia chilensis plants, an endemic berry in Chile. Our approach was to use fluridone, an ABA biosynthesis inhibitor, and then subsequent ABA applications to young and fully‐expanded leaves of drought stressed A. chilensis plants during 24, 48 and 72 h of the experiment. Plants were harvested and leaves were collected separately to determine the biochemical status. We observed that fluridone treatments significantly decreased ABA concentrations and total anthocyanin (TA) concentrations in stressed plants, including both young and fully‐expanded leaves. TA concentrations following fluridone treatment were reduced around fivefold, reaching control plant levels. ABA application restored ABA levels as well as TA concentrations in stressed plant at 48 h of the experiment. We also observed that TA concentrations followed the same pattern as ABA concentrations in the ABA treated plants. Quantitative real‐time PCR revealed that AcUFGT gene expression decreased in fully‐expanded leaves of stressed plants treated with fluridone, while a subsequent ABA application increased AcUFGT expression. Taken together, our results suggest that ABA is involved in the regulation of anthocyanin biosynthesis under drought stress.     

Inhibitory Action of Phenolic Compounds on Abscisic Acid-Induced Abscission

Trans-cinnamic acid, coumarin, salicylic acid, resorcinol, gallicacid, tannic acid and rutin counteracted the abscission-inducingeffect of ABA. This inhibitory action of phenolic compoundson ABA-induced abscission may indicate that they have a regulatoryrole in the process.     

Dormancy and seasonal changes of plant growth regulators in hazel buds

Levels of indole-3-acetic acid (IAA), abscisic acid (ABA) and total phenolic compounds have been determined in hazel ( Corylus avellana L. cv. Negreta) buds. IAA and ABA were quantified by flame ionization detector gas-chromatography, and the phenolic content determined by a colorimetric technique. The highest level of free ABA occurs in autumn, approximately at the onset of winter dormancy, and it is lowest just before bud burst; suggesting that ABA plays an important role in the induction and maintenance of winter dormancy. The opposite result was obtained for IAA; the level of this regulator was low in autumn and showed a sharp increase just before budbreak. With respect to phenols, an apparent correlation can be observed between them and ABA in at least some of the fractions studied (acid and residual), so that they may enhance the inhibitory effect of ABA. The IAA/inhibitor balance showed a clear correlation with dormancy. In autumn (dormant buds), this balance is in favour of the inhibitors while the balance shifts towards IAA at the end of winter.     

Effect of phenolic compounds on abscisic acid-induced stomatal movement: Structure – activity relationship

Application of abscisic acid (ABA) brings about stomatal closure within 30 min in epidermal peels of Vicia faba . A number of phenolic compounds antagonise the effect of ABA. Derivatives of benzoic acid, cinnamic acid, coumarin and flavonoids have been studied in order to establish structure – activity relationship. Derivatives of benzoic acid reverse the ABA effects. Coumarin, esculetin and three hydro derivatives of cinnamic acid fail to show the anti-ABA activity. Thus, the presence of parahydroxyl group and double bond in the side chain is necessary for anti-ABA activity.     

Modulation by phenolic compounds of ABA-induced inhibition of mustard (Brassica juncea L. cv. RLM 198) seed germination

Exogenous abscisic acid (ABA) inhibited the germination of B. juncea seeds in a concentration dependent manner. As revealed in a time-course study, the ABA-induced inhibition got progressively alleviated with the lapse of time following ABA treatment possibly due to metabolic conversion of applied ABA in the seed tissue. A simultaneous application of certain phenolic compounds namely, p-coumaric-, vanillic-, gallic-, and chlorogenic acid (but not caffeic acid) also caused an alleviation of ABA effect. Of the above two patterns of recovery, the phenolic-dependent alleviation of ABA effect was apparent much earlier (24-48 hr treatment) than the time-dependent one (72 hr). It is likely that phenolics could accelerate ABA metabolism in the seed tissue leading to an early recovery from ABA-induced inhibition.     

Effects of Abscisic Acid on Phenolic Content and Lignin Biosynthesis in Tobacco Tissue Culture

A significant depression of callus growth resulted from low concentrations of abscisic acid (ABA) added to the medium recommended by Linsmaier and Skoog. Low concentrations also decreased the chlorogenic acid and lignin content of the callus, and generally decreased amounts of scopolin and scopoletin in the tissue. Gibberellic acid (GA₃) stimulated callus growth in a low concentration (0.1 mg/1) and inhibited growth at a high concentration (10.0 mg/1). Both levels of GA₃ increased scopoletin accumulation in tobacco callus. A high concentration of GA₃ increased the accumulation of scopolin and chlorogenic acids, whereas a low concentration decreased the amounts of these two phenolic compounds. In comparison with the control, lignin synthesis was stimulated by a low GA₃ concentration, but a high GA₃ concentration did not have a significant effect. Both low and high concentrations of GA₃ overcame ABA inhibition of growth and lignin synthesis, and partially reversed ABA inhibition of scopoletin production. However, GA₃ did not reverse the inhibitory effect of ABA on scopolin production. The low concentration of GA₃ overcame the inhibition of chlorogenic acid production resulting from a 0.01 mg/1 concentration of ABA, but this was the only reversal of chlorogenic acid inhibition resulting from addition of GA₃ to the medium.