Regulation of phenylalanine ammonia-lyase genes in carrot suspension cultured cells

Induction of anthocyanin synthesis occurs during metabolic differentiation in carrot suspension cultured cells grown in medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), and is closely correlated with embryogenesis. Anthocyanin synthesis may also be induced by light-irradiation under different culture conditions. The phenylalanine ammonia-lyase (PAL) gene (TRN-PAL), which was transiently induced by the transfer effect, was also rapidly induced after light-irradiation. However, TRN-PAL was not involved in anthocyanin synthesis. A second PAL gene, ANT-PAL, was involved in anthocyanin synthesis. ANT-PAL was induced during metabolic differentiation in medium lacking 2,4-D parallel with the induction of chalcone synthase (CHS). PAL genes in the carrot genome are expressed differentially depending on the nature of the environmental stimulus, e.g. transfer effect and light, and other parameters which also affect anthocyanin synthesis.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - Luc firefly luciferase - PAL phenylalanine ammonia-lyase - UV ultraviolet     

Changes in activities of enzymes involved in flavonoid metabolism during the initiation and suppression of anthocyanin synthesis in carrot suspension cultures regulated by 2,4-dichlorophenoxyacetic acid

When anthocyanin synthesis was induced in cell suspension cultures of carrot ( Daucus carota L. cv. Kurodagosun) by transfer to medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), chalcone synthase (CHS, EC 6.-.-.-), and chalcone-flavanone isomerase (CHFI, EC 5.5.1.6) activities appeared, reaching maxima 6–7 days after transfer. The maximum specific activity of CHS was much lower than that of PAL or CHFI. In a medium containing 2,4-D, no anthocyanin was synthesized, PAL and CHFI activities were suppressed and CHS activity could not be detected at all. The activities of PAL and CHS in cells cultured without 2,4-D for 6 days began to decrease within 3–6 h of 2,4-D addition. CHS activity was completely repressed 24–36 h after the addition, but CHFI activity was almost unchanged at this time. After culture without 2,4-D for 6 days, cell suspensions were transferred to fresh media either lacking or containing 2,4-D. After transfer, PAL increased in both media within 3 h, whereas CHS activity and anthocyanin accumulation were coordinated and both were completely regulated by 2,4-D. Changes in CHS activity rather than PAL activity correlate with changes in anthocyanin accumulation under various culture conditions.     

Changes in Activities of Enzymes Involved in General Phenylpropanoid Metabolism during the Induction and Reduction of Anthocyanin Synthesis in a Carrot Suspension Culture as Regulated by 2,4-D

The activities of enzymes involved in general phenylpropanoidmetabolism were followed in a carrot suspension culture duringthe induction and reduction of anthocyanin synthesis regulatedby 2,4-D. When no anthocyanin synthesis occurred in a mediumcontaining 2,4-D (+2,4-D medium), the activities of phenylalanineammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL) increased1 day after transfer due to the transfer effect, but subsequentlydecreased and remained at a low level. Cinnamate-4-hydroxylase(C4H) activity showed a low level throughout culture. When cellswere transferred to a medium lacking 2,4-D (–2,4-D medium),the activities of PAL, C4H and 4CL increased and maximum activitiesof these enzymes were observed 6–7 days after transfer,when anthocyanin was most rapidly synthesized. When cells were cultured in the –2,4-D medium, the additionof 2,4-D immediately reduced the induced activity of PAL. PALactivity was super-induced by the transfer effect, while anthocyaninsynthesis decreased. The addition of intermediates of generalphenylpropanoid metabolism, with 2,4-D, to the medium 6 daysafter transfer to the –2,4-D medium did not promote anthocyaninsynthesis, whereas dihydroquercetin did promote it. Regulationof anthocyanin synthesis by 2,4-D is discussed in relation tochanges in enzyme activities involved in general phenylpropanoidmetabolism. ¹ Present address: Cell Science and Technology Division, FermentationResearch Institute, Agency of Industrial Science and Technology,Yatabe-machi, Ibaraki 305, Japan. ² Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai 980, Japan.     

Effects of Growth Regulators on the Induction of Anthocyanin Synthesis in Carrot Suspension Cultures

The effects of plant growth regulators were investigated onanthocyanin synthesis induced by removing auxin from carrotsuspension cultures. Of the auxins tested, 2,4-D showed thestrongest inhibiting effect on anthocyanin synthesis and hadthe strongest promoting effect on undifferentiated growth. When2,4-D was added to anthocyanin synthesizing cells, in whichcell division had ceased, anthocyanin synthesis was repressedimmediately, accumulated anthocyanin disappeared and cell divisionresumed. All cytokinins examined promoted anthocyanin synthesisin the absence of auxin. Both gibberellic acid (GA₃) and abscisicacid inhibited anthocyanin synthesis in media lacking 2,4-D,though GA³ showed no effect on cell division. These effectsof growth regulators on anthocyanin synthesis are similar tothose reported for their effects on embryogenesis [Fujimuraand Komamine (1975) Plant Sci. Lett. 5: 359, (1979) Z. Pflanzenphysiol.95: 13, (1980) Z. PJlanzenphysiol. 99: 1]. The relationshipbetween the induction of anthocyanin synthesis, metabolic differentiation,and embryogenesis are discussed. ¹ Present address: Department of Biology, College of Arts andSciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo153, Japan. ² Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai, Miyagi 980, Japan. (Received November 28, 1985; Accepted July 23, 1986)     

Anthocyanin accumulation and changes in activities of phenylalanine ammonia-lyase and chalcone synthase in roselle (Hibiscus sabdariffa L.) callus cultures

Time-course changes in anthocyanin accumulation, phenylalanine ammonia-lyase activity and chalcone synthase activity were examined in roselle callus tissues incubated under different culture conditions. Phenylalanine ammonia-lyase activity was not affected by either the kind of auxin supplemented to the medium or light regime. In contrast, chalcone synthase activity was markedly suppressed when the callus was cultured with a medium containing indole-3-acetic acid instead of 2,4-dichlorophenoxyacetic acid (2,4-D) or in the dark. The results imply that in roselle callus cultures chalcone synthase plays a more important role in anthocyanin biosynthesis regulated by 2,4-D and light irradiation than phenylalanine ammonialyase.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - PAL phenylalanine ammonia-lyase - CHS chalcone synthase     

Light-Induced Synthesis of Anthocyanin in Carrot Cells in Suspension: I. THE FACTORS AFFECTING ANTHOCYANIN PRODUCTION

Takeda, J. 1988. Light-induced synthesis of anthocyanin in carrotcells in suspension. I. The factors affecting anthocyanin production.—J.exp. Bot. 39: 1065–1077. A light-triggered anthocyanin-synthesizing system was establishedfor carrot cells in suspension. A few days after transfer ofthe cells to a 2,4-dichlorophenoxyacetic acid (2,4-D)-free mediumin the dark, light irradiation triggered anthocyanin synthesisand concomitantly stopped expansion growth. Over 90% of thecells synthesized anthocyanin without cell division. By loweringthe concentration of phosphate or both nitrogen and phosphateand delaying the time of onset of irradiation, the productionof anthocyanin per cell increased to a maximum level of 0–8µmol anthocyanin per 10⁶ cells. A change in the physiologicalstate of cells (light-insensitive to light-sensitive state)induced by the transfer to 2,4-D-free medium is suggested tobe a prerequisite for the light-triggered synthesis of anthocyanin. Key words: Anthocyanin production, cultured cells, Daucus carota, light-triggered, 2,4-D     

Induction of anthocyanin pigments in callus cultures of <Emphasis Type="Italic">Rosa hybrida</Emphasis> L. in response to sucrose and ammonical nitrogen levels

The influence of varied concentrations of sucrose and ammonical (NH₄⁺) nitrogen on in vitro induction and expression of anthocyanin pigments from Rosa hybrida cv. ‘Pusa Ajay’ was investigated. Of two explants (petal and leaf discs) selected and cultured under two different conditions (light and dark), leaf discs were found to be most suitable for callus initiation. Profuse and early callus induction was observed when leaf discs of rose were cultured under total dark conditions on solid Murashige and Skoog (MS) medium supplemented with 4.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Early pigment initiation, enhancement and maximum anthocyanin production from calluses were recorded when leaf discs were cultured on Euphorbia millii (EM) medium supplemented with 7% sucrose compared with calluses cultured at 4% sucrose concentration under 16/8 h (light/dark) photoperiod regime. Reducing the concentration of NH₄⁺ nitrogen in the solid MS medium led to slight improvement in anthocyanin production in rose leaf calluses.     

Characterization of Intravacuolar Pigmented Structures in Anthocyanin-Containing Cells of Sweet Potato Suspension Cultures

Intravacuolar pigmented structures occurred in anthocyanin-producingcultured cells of sweet potato (Ipomoea batatas) were characterized.Formation of the pigmented structures in sweet potato cellswas induced by transfer of callus cultured in 2,4-D containingagar medium into 2,4-D free liquid medium under continuous illumination.These structures were found in the vacuoles. The pigmented structureswere isolated from the protoplasts by precipitation in 60% (w/w)sucrose after centrifugation. Electron microscopic observationsof the anthocyanin-containing cultured cells showed these structureshad neither membrane boundary nor internal structures, and werefound as strongly osmiophilic globules in vacuoles. Numeroussmall osmiophilic globules were observed in central vacuolesat the early stage of anthocyanin accumulation, but not foundin cytoplasm. Similar pigmented structures in vacuoles werealso formed by treatment with neutral red. These observationsindicate that these pigmented structure is the high densityand insoluble globules highly concentrated with anthocyanin,which was synthesized in cytoplasm and transported to the centralvacuoles. ⁴Present address: Department of Cell Biology, National Institutefor Basic Biology Myodaijicho, Okazaki, 444 Japan     

Studies on Foliar Penetration: I. FACTORS CONTROLLING THE ENTRY OF 2, 4 - DICHLOROPHENOXYACETIC ACID

A technique, using leaf disks, has been developed to study thepenetration of isotopically labelled compounds into leaves underconditions where there is no appreciable change in the concentrationof the external solution and no subsequent translocation. Inthis preliminary survey, the leaves of Phaseolus vulgaris andColeus Blumei were employed to investigate the entry of 2,4-dichlorophenoxyaceticacid (2,4-D), labelled in the carboxyl group with ¹⁴C. Over3 days there is no loss of ¹⁴C to the atmosphere from treatedleaves of Phaseolus. The rate of penetration is enhanced when(a) the leaves are young, (b) the water status is lowered, (c)the temperature is raised (Q₁₀=2.3–2.8), and (d) a surface-activeagent is added to the external solution. Penetration is alsofavoured by a decrease in the pH, the relation indicating thatboth ions and molecules enter. Penetration is greater in thelight and prior illumination of the tissues positively affectsthe subsequent rate in the light, but not in the dark. In boththe light and the dark considerably more 2,4-D penetrates theabaxial surface of Phaseolus leaves. For Coleus an even greaterdifference between surfaces is found in the light but not inthe dark. For both species in the light the rates of entry intoboth surfaces are proportional to their respective stomataldensities. The simultaneous addition of indoleacetic acid tothe external solution caused more 2,4-D to enter Phaseolus leaves,but the addition of triiodobenzoic acid restricts entry. Therate of penetration remains constant over 24 hours and between0.1 and 200 mg./l. the rate is linearly related to concentration.Subsequent to entry, the 2,4-D is in a form which does not diffusefrom the tissue into buffer or exchange with unlabelled 2,4-D.Moreover, no outward movement takes place from treated tissuewhich has been frozen and thawed. These findings are discussedin relation to previous work on foliar penetration. It is concludedthat at least with Phaseolus penetration largely takes placethrough the guard cells and/or accessory cells.     

Studies on Foliar Penetration: 2. THE ROLE OF LIGHT IN DETERMINING THE PENETRATION OF 2,4 DICHLOROPHENOXYACETIC, ACID

A further study has been made of the factors determining thelevel of penetration of 2,4-dichlorophenoxyacetic acid (2,4-D)into leaves. The technique involves the use of leaf disks and2,4-D containing carbon-14 in the carboxyl group. For Phaseolus vulgaris the influence of the pH of the appliedsolution is greater in the light than in the dark. Between 0and 1,000 f.c. at 27° C there is a small increase in therate of penetration into the abaxial surface. Under these conditionsthe rates remain constant up to 56 hours. This linear relationshipholds for concentrations ranging from 100 to 1,000 mg/l, andthe rate of penetration is directly proportional to the concentration.For intensities in excess of 1,000 f.c. the light response ismarkedly different: over the first few hours there is a steadyand relatively slow rate of penetration which is followed bya second phase when the rate is greatly accelerated. This acceleratedrate can be reversed by transferring the disks to darkness,and does not take place at 1° C. Likewise, if excised disksare left for more than one hour in either the light or the darkbefore applying 2,4-D then there is subsequently no phase ofaccelerated penetration. The course of penetration into theadaxial surface exhibits no accelerated rate, and compared tothe abaxial surface the rates are lower. For leaves of Ligustrum ovalifolium, which lack stomata on theadaxial surface, the rates of penetration at 27° C intoboth surfaces remain constant in either light or darkness. Forboth the adaxial and abaxial surfaces, the rate progressivelyincreases from 0 to; 2000 f.c. and there is no phase of acceleratedpenetration. Penetration into the adaxial surface is less. At1° C the rates for both surfaces in either light or darknessare depressed. If disks of Phaseolus are irradiated with ultraviolet light,subsequent penetration is markedly depressed in the light at27° C, but in the dark or at 1° C it is enhanced. On the basis of these findings it is concluded that both physicaland metabolic factors control the rate of penetration of 2,4-DTransport through the cuticle will be dependent on adsorptionand the length of the diffusion paths. Once diffusion gradientshave been established between the surface of the cuticle andthe outer surface of the cytoplasm in the epidermal cells, thesteepness will be dependent on the rates at which 2,4-D is eitherconverted into some metabolite or moved away from the surfaceor into other cells. The relative importance of physical andmetabolic process will be dependent on the permeability andthickness of the cuticle and the level of metabolic activity.The possible role of ectodesmata in determining both the lengthand steepness of the diffusion paths is discussed.     

Generation of Seakale (Crambe maritima L.) Plantlets by Tissue Culture

When placed in shaken liquid culture in MS medium, callus derivedfrom petioles of seakale (Crambe maritima L.) failed to fragmentto produce a cell suspension culture. In the absence of 2,4-D,growth was very good, and hollow, dark green, trumpet-shapedbodies were produced, but in the presence of 2,4-D, solid, palergreen pear-shaped bodies were formed. When grown on filter paperbridges with White's medium, a small proportion of both typesof bodies underwent slow, partly-controlled development culminating,after several months, in the formation of plantlets. Seakale, Crambe maritima L., in vitro propagation, callus culture     

Studies on the Growth in Culture of Plant Cells: XXIV. EFFECTS OF 2, 4-DICHLOROPHENOXYACETIC ACID AND LIGHT ON THE GROWTH AND METABOLISM OF ACER PSEUDOPLATANUS L. SUSPENSION CULTURES

Omission of 2,4-dichlorophenoxyacetic acid (2,4-D) from batchcultures of sycamore (AcerpseudoplatanusL.) caused growth cessationafter an initial period of exponential growth. The presenceof white light reduced the amount of growth after 2,4-D withdrawal.Growth cessation, in the absence of 2,4-D, was accompanied bythe accumulation of the free amino acid, serine. This accumulationbegan before the cessation of growth and was rapidly reversedby the re-addition of 2,4-D.     

The Uptake of Growth Substances: XV. THE DIFFERENTIAL EFFECTS OF PROGRESSIVE CHLORINATION OF PHENOXYACETIC ACID ON ENTRY INTO THE EPIDERMAL AND CUT SURFACES OF STEM SEGMENTS

The comparative patterns of entry into segments with sealedand open ends, excised from etiolated internodes of Pisum sativum,have been examined for phenoxyacetic acid (POA) and its 2-,4-, 2,4-, 2,6-, 3,5-, 2,4,5- and 2,4, 6-chloro derivatives,each containing ¹⁴C in the carboxyl group. Sealing the ends greatly depresses the level of entry, on averagean eight-fold reduction at 9 h. Likewise, the interrelationsbetween the degree of chlorination and uptake potential aredisparate. For segments with exposed cut surfaces the finalcontent is maximal for POA and the 2-chloro compound and minimalfor the 3,5-dichloro derivative (3,5-D) with an eight-fold difference.With sealed ends this difference is reduced to two-fold butwhile 3,5-D accumulates least uptake is now highest for POAand 2,4-D. There are also changes in the order with time. Initially,2,4,5-T penetrates fastest into sealed segments but for segmentswith open ends entry is most rapid for the 4- and 2,4,6-chloroderivatives. Additions of streptomycin and cetyltrimethylammoniumbromide(CTAB) induce differential changes in the patterns of uptake.Where uptake is promoted the enhancement is not restricted toactive auxins. Sealing the ends may alter the nature of theresponse. The likely physico-chemical and metabolic processes concernedin the two routes of entry are discussed and the results comparedwith previous divergent findings on the relationship betweenchemical structure and uptake by Lemna minor and penetrationinto leaves of Phaseolus vulgaris.     

The Regulation of Synthesis of Phenolics in Stationary Phase Cell Cultures of Acer pseudoplatanus L.

The addition of 2/ (w/v) sucrose to stationary phase suspensioncultures of sycamore (Acer pesudoplatanus) in medium depletedof nitrogen and phosphorus leads to the accumulation of highlevels of phenolics after a 5 d lag period. Urea and 2,4-D inhibitthis accumulation of phenolics. Urea results in a stimulationin cell protein content coincident with phenolics in hibition,and leads to a substantial diversion of the common precursorphenylalanine from phenolics synthesis into protein synthesis.2,4-Dichlorophenoxyacetic acid (2,4-D) does not lead to proteinaccumulation since the cultures are nitrogen-limited, but stimulatesan increase in protein turnover coincident with phenolics inhibition.The role of protein synthesis in the regulation of phenolicsmetabolism through competition for common precursors is discussed.     

The effect of light and 2,4-D on anthocyanin production in Oxalis reclinata callus

In vitro cultures of O. reclinata accumulate red anthocyanin pigments. Two callus lines were established from O. reclinata, one red and the other non-pigmented. The red callus accumulated cyanidin-3-glucoside as a major pigment. Light irradiation induced anthocyanin synthesis in white callus, resulting in a heterogenous red callus line being formed. The incubation of red and white callus cultures in the dark or at low-light resulted in the repression of red pigment accumulation. The application of 2,4-D (1.0 mg l^-1) inhibited pigment production in the white callus and decreased anthocyanin accumulation in the red callus. The polypeptide composition of the red and white callus lines from O. reclinata were compared using two-dimensional electrophoresis. The red callus had a larger subset of neutral and acidic polypeptides.