Photocontrol of anthocyanin formation in turnip seedlings

Summary The substitution of red or blue light for the first six hours of prolonged irradiation with far-red light reduced anthocyanin formation by about 60%; red or far-red light similarly substituted for blue light had little effect. It is concluded that the effects of prolonged irradiation with blue and far-red depend, in part at least, on different photoreceptors.The effects of pre-treatment with red or blue light also occurred when only short exposures to light were given, and were reversed by immediate brief exposures to far-red. The depressing effect of a short pre-irradiation treatment was largely prevented if seedlings were kept at low temperature or in an atmosphere of nitrogen in the dark period before transfer to the prolonged far-red treatment. The effect of the pre-irradiation treatment is attributed to enzymatic destruction of phytochrome following conversion to the P_FR form, and it is suggested that anthocyanin synthesis in far-red light largely depends on phytochrome, possibly due to the maintenance of a low level of P_FR in the tissue by the absorption tail of P_R in the far-red.A pre-irradiation treatment with red also decreased the inhibitory effect of far-red on hypocotyl elongation but did not change the response to blue light.

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Zusammenfassung Die Anthocyanbildung war im langfristig gegebenen Dunkelrot bis zu etwa 60% reduziert, wenn die ersten 6 Std durch hellrote oder blaue Bestrahlung ersetzt wurden; Hellrot oder Dunkelrot in gleicher Weise im Dauerblaulicht substituiert waren praktisch wirkungslos. Daraus wird geschlossen, daß der Effekt einer Dauerbestrahlung mit Blau und Dunkelrot zum Teil jedenfalls, auf verschiedene Photorezeptoren zurückzuführen ist.Der Effekt einer Vorbehandlung mit hellrotem oder blauem Licht trat auch dann auf, wenn nur kurzfristige Bestrahlungen gegeben wurden und konnte durch unmittelbar nachfolgende kurze Dunkelrot-Belichtung wieder aufgehoben werden. Die Hemmung durch kurzfristige Vorbestrahlung konnte weitgehend verhindert werden, wenn die Keimlinge während der Dunkelperiode, vor der Übertragung in Dauerdunkelrot, bei tiefer Temperatur oder unter Stickstoff gehalten wurden. Der Vorbelichtungseffekt wird auf die enzymatische Destruktion von Phytochrom, nach der Umwandlung in die P_FR-Form, zurückgeführt und es wird vermutet, daß die Anthocyansynthese im Dauerdunkelrot weitgehend phytochromabhängig ist, wahrscheinlich durch die Aufrechterhaltung eines niedrigen P_FR Niveaus im Gewebe infolge der schwachen Absorption von P_R im Dunkelrot.Eine Vorbelichtung mit Hellrot verringerte ebenfalls die hemmende Wirkung von Dunkelrot auf das Hypokotylwachstum, war jedoch ohne Einfluß im Blaulicht.

     

Photocontrol of anthocyanin formation in turnip seedlings

Summary The insertion of a dark period during irradiation with far-red resulted in the occurrence of a second lag-phase in the synthesis of anthocyanin when the seedlings were returned to light. The length of this second lag-phase was similar, to the first. It is suggested that the action of far-red light may not involve gene activation but rather the formation of substrate(s); the lag-phase may arise from ratelimiting steps in the synthesis of anthocyanin pigment.     

Photocontrol of anthocyanin formation in turnip seedlings

Summary When irradiated for a period of 48 hours with light of restricted wave-lengths in the red and far-red regions, maximum anthocyanin content in hypocotyls was found at 730 nm; in cotyledons the yield at 716 was almost equal to that at 730 nm.In hypocotyls the effectiveness of 703, 716 and 760 nm light was much increased if plants were transferred to red light after 6 hours. The intermediate wave-length band centred at 703 nm was ineffective when given alone but led to a high yield if followed by red light. When given before far-red light (standard far-red field), 6 hours at 703 nm reduced anthocyanin yield in the same way as red light; when given after 6 hours of far-red, however, 703 nm light did not act like red light to sustain a high rate of anthocyanin synthesis.It is concluded that two reactions are involved in the photocontrol of anthocyanin formation in turnip seedlings.     

Contributions of photosynthesis and phytochrome to the formation of anthocyanin in turnip seedlings

Turnip seedlings (Brassica rapa L.) irradiated for 24 hours with radiation at 720 nanometers synthesize chlorophyll a and anthocyanin. Antimycin A and 2,4-dinitrophenol, which are known to reduce cyclic photophosphorylation, also reduce anthocyanin synthesis. Noncyclic photophosphorylation is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and o-phenanthroline. These compounds promote cyclic photophosphorylation and anthocyanin synthesis. On the basis of these findings it is suggested that the photomorphogenic response of anthocyanin synthesis in turnip seedlings arises in part through photosynthetic activity.     

Action of light and streptomycin on protein synthesis in cabbage seedlings

The action of light on protein synthesis was examined in the cabbage seedlings, a system extensively used in the studies of anthocyanin synthesis. Continuous red and far red light have no effect on total protein content while they cause a marked decrease in the level of free amino acids in cabbage seedlings. The rate of protein synthesis, measured as incorporation of radioaetively-labelled amino acids into proteins, is clearly stimulated by light. Phytochrome involvement in the light stimulation of the incorporation is also demonstrated by the red-far red reversibility of the response. The relative effectiveness of continuous red and far red light upon the incorporation of amino acids into proteins is affected by the nature of the system used to study the incorporation process. When excised cotyledons and short period of incorporation were used, continuous far red was more effective than red. However, when whole seedlings and long period of incorporation were used, red and far red were equally effective. Streptomycin causes a 10– 15% decrease in the rate of incorporation of amino acids into proteins of all cellular fractions, except the plastid fraction where a much higher inhibition (30%) was observed.     

Effectiveness of intermittent light treatments on anthocyanin synthesis in dark-grown and light-pretreated seedlings

Differences in the extent of anthocyanin production between intermittent light treatments with short and long dark intervals between successive irradiations are more pronounced in dark-grown than in light-pretreated cabbage seedlings. This observation is consistent with the hypothesis, based on destruction kinetics data, that there might be two pools of phytochrome, a labile one and a stable one, present in different proportions in dark-grown and light-pretreated seedlings, and suggests that light-dependent changes of the stable to labile phytochrome ratio might be physiologically significant in the photoregulation of photomorphogenic responses.     

Comparison of the action of light on ribosomal RNA synthesis in cabbage and mustard seedlings

We have compared the action of light on ribosomal RNA synthesis in mustard and cabbage seedlings, two of the most frequently used systems for the studies of anthocyanin synthesis. The level of RNA (both t-RNA and r-RNA) “stored” in mustard dry seeds is much lower than in cabbage dry seeds. The kinetics of RNA synthesis in cabbage and mustard seedlings exposed to light are very different: In cabbage seedlings, light produces no apparent stimulation of cytoplasmic r-RNA synthesis, while it does increase plastid r-RNA synthesis. On the other hand, in mustard seedlings, light promotes both cytoplasmic and plastid ribosomal RNA synthesis. Streptomycin, which inhibits chlorophyll formation and chloroplast development while having no effect (mustard) or enhancing (cabbage) anthocyanin synthesis in these two systems, is in both cases an effective inhibitor of plastid r-RNA synthesis, but not of cytoplasmic r-RNA synthesis.     

Genetical study on the formation of anthocyanins and flavonols in turnip varieties. Genetical studies on anthocyanins in brassicaceae II

Genetic studies were made on the root color of turnip varieties. In the cross between the red (raphanusin, pelargonidin glucoside) and the white varieties, F₁ hybrids were all purple (rubrobrassicin, cyanidin glucoside), and F₂ progenies were segregated into three groups: purple (rubrobrassicin), red (raphanusin), and white in the ratio of 9:3:4, respectively. This indicates the presence of two pairs of conditional alleles, as previously found in the case of radish. As to the flavonols in turnip varieties, isorhamnetin and kaempferol were found in the plants forming rubrobrassicin in roots, and only kaempferol in the plants producing raphanusin in roots; whereas isorhamnetin was found together with a trace of kaempferol in the plants with white root.     

Photocontrol of the germination of onoclea spores: I. Action spectrum

Light stimulates the germination of spores of the fern Onoclea sensibilis L. At high dosages, broad band red, far red, and blue light promote maximal germination. Maximal sensitivity to these spectral regions is attained from 6 to 48 hours of dark presoaking, and all induced rapid germination after a lag of 30 to 36 hours. Maximal germination is attained approximately 70 hours after irradiation. Dose response curves suggest log linearity. The action spectrum to cause 50% germination shows that spores are most sensitive to irradiation in the red region (620-680 nm) with an incident energy less than 1000 ergs cm⁻²; sensitivity decreases towards both shorter and longer wavelengths. Although the action spectrum is suggestive of phytochrome involvement, photoreversibility of germination between red and far red light has not been demonstrated with Onoclea spores. An absorption spectrum of the intact spores reveals the presence of chlorophylls and carotenoids. Since the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea does not inhibit germination, it is concluded that photosynthesis does not play a role in the germination process.     

The biogenesis of the anthocyanins. III. The role of sugars in anthocyanin formation

Anthocyanin formation in growing cultures of Spirodela is promoted by sucrose but not by glucose; conversely, growth is promoted by glucose but not by sucrose. Fructose is intermediate in both respects.In non-growing cultures, however, all three sugars are equally effective in promoting anthocyanin formation.A number of treatments which increase or decrease the anthocyanin content have parallel effects on the reducing sugar content. A plot of anthocyanin content against reducing sugar content shows a smooth relationship. Variations in the sucrose content are smaller and show no parallelism with pigmentation.It is deduced that anthocyanin may be formed independently from any of the three sugars, but that glucose is preferentially consumed for growth.Phosphate apparently does not participate in the formation of anthocyanin, and if the process does take place directly from sugars it probably does not proceed via the usual glycolytic pathway, since none of a number of glycolytic intermediates gives rise to any anthocyanin.There is some evidence for a participation of meso-inositol in the biogenesis of anthocyanin. The effects of quinic and shikimic acids appear too small for them to be considered as intermediates. Phosphate appears to be no more required with inositol than with the sugars.     

Effect of temperature on phytochrome—mediated anthocyanin synthesis in red cabbage

Abstract. A brief high temperature treatment (45 C) promoted anthocyanin synthesis in 2-d-old dark-grown red cabbage seedlings. The increased effectiveness of a temperature/red light treatment as opposed to the reverse sequence suggests that an elevated temperature 'induces' some component which facilitates the phytochrome response.     

Effect of chlorocholine chloride and gibberellic acid on the anthocyanin synthesis in radish seedlings

Anthocyanin synthesis in radish ( Raphanus sativus L. cv. Scarlet Globe) seedlings after treatment with chlorocholine chloride (CCC) and gibberellic acid (GA) has been investigated. CCC promotes and GA₃ inhibits the synthesis. When both substances are given together, CCC reverses the inhibition caused by GA₃. Simultaneous external feeding of anthocyanin precursors (sucrose and phenylalanine) reverses the GA₃ inhibition. A higher amount of total free amino acids, in particular phenylalanine, was present in CCC-treated seedlings compared to controls grown on distilled water. The amount of phenylalanine was lower in seedlings treated with both CCC and GA₃ as compared to seedlings treated with CCC alone, and total free sugars (reducing plus non-reducing) was lower in CCC treated seedlings than in controls grown on distilled water. We conclude that CCC and GA3 control the anthocyanin synthesis at the level of precursors.     

Red-far red reversibility of anthocyanin synthesis in dark-grown and light-pretreated seedlings

The rate of escape from red—far red reversibility for anthocyanin synthesis is lower in dark-grown than in light-pretreated seedlings of Brassica oleracea L. Red Acre, Secale cereale L. Cougar, and Lycopersicon esculentum Mill. Beefsteak. This observation is consistent with the suggestion that there might be two pools of phytochrome, a labile one, characterized by fast disappearance of phytochrome—far-red absorbing form, and a stable one, characterized by slow disappearance of phytochrome—far-red absorbing form, and that the proportion between the two pools might be different in dark-grown and light-pretreated seedlings.     

Pattern formation underlying phytochrome-mediated anthocyanin synthesis in the cotyledons ofSinapis alba L.

The ability to respond to phytochrome (P_fr, the far-red light absorbing from of phytochrome) with anthocyanin synthesis appears first in some marginal regions of the abaxial epidermis of the mustard cotyledons and from there spreads gradually over the entire tissue (transient phase). The pertinent pattern is independent of environmental influences such as light quality and nutritional culture conditions. The competence for P_fr in the epidermal cells, with regard to the initial action of P_fr (concerning anthocyanin synthesis), appears considerably earlier than the ability for actual anthocyanin synthesis. An electron microscopical study of the ultrastructural changes occurring in vacuoles and plastids of the epidermal cells during the transient phase showed that a correlation only exists between the differentiation of central cell vacuoles, originating from the aleurone vacuoles, and the appearance of the ability to accumulate anthocyanin. It is suggested that the formation of a central cell vacuole is a prerequisite for anthocyanin accumulation in the epidermal cells of the mustard seedling cotyledons.Abbreviations P_r, P_fr red and far-red absorbing forms of phytochrome - HS Hoagland's nutrient solution     

Action of light,nitrate and ammonium on the levels of NADH- and ferredoxin-dependent glutamate synthases in the cotyledons of mustard seedlings

In mustard (Sinapis alba L.) cotyledons, NADH-dependent glutamate synthase (NADH-GOGAT, EC 1.4.1.14) is only detectable during early seedling development with a peak of enzyme activity occurring between 2 and 2.5 d after sowing. With the beginning of plastidogenesis at approximately 2 d after sowing, ferredoxindependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) appears while NADH-GOGAT drops to a very low level. The enzymes were separated by anion exchange chromatography. Both enzymes are stimulated by light operating through phytochrome. However, the extent of induction is much higher in the case of Fd-GOGAT than in the case of NADH-GOGAT. Moreover, NADH-GOGAT is inducible predominantly by red light pulses, while the light induction of Fd-GOGAT operates predominantly via the high irradiance response of phytochrome. The NADH-GOGAT level is strongly increased if mustard seedlings are grown in the presence of nitrate (15 mM KNO₃,15 mM NH₄NO₃) while the Fd-GOGAT level is only slightly affected by these treatments. No effect on NADH-GOGAT level was observed by growing the seedlings in the presence of ammonium (15 mM NH₄Cl) instead of water, whereas the level of Fd-GOGAT was considerably reduced when seedlings were grown in the presence of NH₄Cl. Inducibility of NADH-GOGAT by treatment with red light pulses or by transferring water-grown seedlings to NO ₃ ^- -containing medium follows a temporal pattern of competence. The very low Fd-GOGAT level in mustard seedlings grown under red light in the presence of the herbicide Norflurazon, which leads to photooxidative destruction of the plastids, indicates that the enzyme is located in the plastids. The NADH-GOGAT level is, in contrast, completely independent of plastid integrity which indicates that its location is cytosolic. It is concluded that NADH-GOGAT in the early seedling development is mainly concerned with metabolizing stored glutamine whereas Fd-GOGAT is involved in ammonium assimilation.Abbreviations and symbols c continuous - D darkness - Fd-GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1) - FR far-red light (3.5 W·m^-2) - NADH-GOGAT NADH-dependent glutamate synthase (EC 1.4.1.14) - Pfr far-red absorbing form of phytochrome - Ptot total phytochrome - R red light (6.8 W· m^-2) - RG9-light long wavelength FR (10 W·m^-2, _RG9<0.01) - () Pfr/Ptot=wavelength-dependent photoequilibrium of the phytochrome system     

Genetic regulation and photocontrol of anthocyanin accumulation in maize seedlings.

The flavonoid pathway leading to anthocyanin biosynthesis in maize is controlled by multiple regulatory genes and induced by various developmental and environmental factors. We have investigated the effect of the regulatory loci R, B, and Pl on anthocyanin accumulation and on the expression of four genes (C2, A1, Bz1, and Bz2) in the biosynthetic pathway during an inductive light treatment. The results show that light-mediated anthocyanin biosynthesis is regulated solely by R; the contributions of B and Pl are negligible in young seedlings. Induction of the A1 and Bz2 genes by high fluence-rate white light requires the expression of a dominant R allele, whereas accumulation of C2 and Bz1 mRNA occurs with either a dominant or recessive allele at R. A1 and Bz2 mRNA accumulate only in response to high fluence-rate white light, but Bz1 is fully expressed in dim red light. Some C2 mRNA is induced by dim red light, but accumulation is far greater in high fluence-rate white light. Furthermore, expression from both dominant and recessive alleles of the regulatory gene R is enhanced by high fluence-rate white light. Seedlings with a recessive allele at R produce functional chalcone synthase protein (the C2 gene product) but accumulate no anthocyanins, suggesting that, in contrast to the R-mediated coordinate regulation of C2 and Bz1 observed in the aleurone, C2 expression in seedlings is independent of R and appears to be regulated by a different light-sensitive pathway.     

Anthocyanin formation in turnip seedlings (Brassica rapa L.): Evidence for two light steps in the biosynthetic pathway

Summary In turnip seedlings, anthocyanin synthesis can be induced with light as soon as water uptake enables the seed coat to be removed. In very young seedlings the main site of production is in the cotyledons but this moves to the hypocotyl when the period of dark growth, before transfer to the light, is increased. The total amount of anthocyanin formed decreases as the seedlings become older. It is suggested that a substance stored in the cotyledons is needed for anthocyanin synthesis and that this substance disappears during growth in the dark. It cannot be replaced by known anthocyanin precursors such as phenylalanine, acetate, shikimic acid and sugars.Anthocyanin synthesis in the hypocotyl is almost completely prevented when the cotyledons are excised, or covered: no anthocyanin is formed in the hypocotyl when the cotyledons alone are irradiated. Cotyledons that have been excised from the hypocotyl synthesize about as much anthocyanin as is formed in the whole intact seedling, but covering the hypocotyl does not increase the amount formed in the cotyledons. These results suggest that pigment synthesis begins in the cotyledons, where a light reaction is needed for the formation of a precursor; the precursor is translocated to the hypocotyl where a second photochemical reaction is required for anthocyanin synthesis. If translocation to the hypocotyl is prevented, anthocyanin is formed in the cotyledons. The nature of the transported precursor is not yet known.

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Zusammenfassung In Keimlingen vonBrassica rapa kann Anthocyansynthese durch Licht induziert werden, sobald es möglich ist, die Samenschale zu entfernen. In den jüngsten Keimlingen sind die Kotyledonen der Ort stärkster Anthocyanbildung, in älteren Keimlingen das Hypokotyl. Die Gesamtmenge an gebildetem Anthocyan nimmt mit zunehmendem Alter der Keimlinge ab. Es wird vermutet, daß eine für Anthocyansynthese notwendige Substanz in den Kotyledonen gespeichert ist und während des Wachstums im Dunkeln abnimmt. Diese Substanz konnte durch bekannte Anthocyanvorstufen wie Phenylalanin, Acetat, Shikimisäure und Zucker nicht ersetzt werden.Anthocyansynthese ist im Hypokotyl fast vollständig unterdrückt, wenn die Kotyledonen entfernt oder verdunkelt werden: Kein Anthocyan wird im Hypokotyl gebildet, wenn die Kotyledonen allein belichtet werden. Isolierte Kotyledonen synthetisieren ungefähr die gleiche Menge Anthocyan wie intakte Keimlinge, aber eine Verdunkelung des Hypokotyls bewirkt keine Steigerung der Anthocyanbildung in den Kotyledonen. Diese Ergebnisse lassen vermuten, daß die Synthese von Anthocyan in den Kotyledonen beginnt, wo eine lichtabhängige Reaktion zur Bildung einer Zwischenstufe notwendig ist; diese Zwischenstufe wird in das Hypokotyl transportiert, wo eine zweite photochemische Reaktion für Anthocyansynthese erforderlich ist. Wird der Transport in das Hypokotyl verhindert, findet Anthocyansynthese in den Kotyledonen statt. Über die Natur dieser Zwischenstufe ist jedoch noch nichts bekannt.

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With 9 Figures in the Text