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.     

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 Prechill-induced Dark Germination in Sorghum halepense (L.) Pers. Seeds by Phytochrome Transformations

A 10 C dark prechilling of johnsongrass [Sorghum halepense (L.) Pers.] seeds, when terminated by a 2-hr, 40 C temperature shift, potentiates about 40% germination at 20 C in darkness. Irradiation of the seeds before, during, and at the end of prechilling with far red light reduces the subsequent germination, although red irradiation after the far red can overcome some of the inhibition. However, either brief red or far red irradiation given immediately after the temperature shift inhibits subsequent germination by one-third to one-half. The results suggest that the far red-absorbing form of phytochrome is a factor in the prechill-induced dark germination and that phytochrome participates in the inhibition of germination by irradiations immediately after the temperature shift.     

Involvement of phytochrome and a blue light photoreceptor in UV-B induced flavonoid synthesis in parsley (Petroselinum hortense Hoffm.) cell suspension cultures

Flavonoid synthesis in cell suspension cultures of parsley (Petroselinum hortense Hoffm.) occurs only after irradiation with ultraviolet light (UV), mainly from the UV-B (280–320 nm) spectral range. However, it is also controlled by phytochrome. A P_fr/P_tot ratio of approximately 20% is sufficient for a maximum phytochrome response as induced by pulse irradiation. Continuous red and far red light, as well as blue light, given after UV, are more effective than pulse irradiations. The response to blue light is considerably greater than that to red and far red light. Continuous red and blue light treatments can be substituted for by multiple pulses and can thus probably be ascribed to a multible induction effect. Continuous irradiations with red, far red and blue light also increase the UV-induced flavonoid synthesis if given before UV. The data indicate that besides phytochrome a separate blue light photoreceptor is involved in the regulation of the UV-induced flavonoid synthesis. This blue light receptor seems to require the presence of P_fr in order to be fully effective.Abbreviations HIR high irradiance response - P_fr far red absorhing form of phytochrome - P_tet total phytochrome - UV ultraviolet light     

Temperature and photocontrol of onoclea spore germination

Germination of Onoclea sensibilis L. spores is controlled by light and temperature. Temperatures of 30 C can induce maximal germination in the dark to a level of 60 to 95% of that induced by a saturating dose of red light (0.38 joules/square meter) providing the spores are placed at the elevated temperature immediately after being sown. Maximum dark germination occurs with a minimum exposure of 16 to 24 hours at 30 C, suggesting that the temperature treatment is required for the induction of germination rather than for the germination process per se. Interaction of temperature and light for induction of germination shows nonadditive behavior. Germination induced by light and temperature applied consecutively never exceeded that which could be induced by a saturating dose of red light alone. Imbibition of the spores at 25 C in the dark for 12 or more hours prior to incubation at 30 C results in a loss of thermosensitivity. Dose response curves for red light induction of germination after varying times of imbibition at 25 C show no concomitant loss of sensitivity of the spores to red irradiation. This suggests that the mechanism and/or pathway of thermoinduction of germination differs from that of photoinduction. The loss of thermosensitivity as a result of presoaking at 25 C can be prevented if the spores are imbibed at 25 C in osmotic agents such as 0.3 molar mannitol or 0.1 gram per liter of polyethylene glycol 400 or in 0.08% dimethylsulfoxide or 10 micrograms per milliliter of herbicide SAN 9789 (4-chloro-5-(methylamino)-2-(α,α,α-trifluoro-m-tolyl-3-(2H)pyridazinone). The latter two substances are hypothesized to act upon membranes. These results suggest that the degree of hydration and possibly changes in membrane properties play a role in the change in sensitivity of Onoclea spores to 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.     

Effect of Temperature on Phytochrome-mediated Responses in Seedlings of Mustard

Anthocyanin synthesis, hair formation, and the synthesis of ascorbic acid oxidase are all phytochrome-mediated reactions occurring in the hypocotyl of mustard (Sinapis alba L.). An investigation was conducted into the effects of temperature on the light promotion of these three photoresponses. When given prior to a light exposure there was either no difference between a 20 and 30 C temperature treatment, or the 30 C treatment resulted in a greater photoresponse. When given subsequent to a light exposure 20 C was far more effective in promoting the responses than was a 30 C treatment. These results are in accordance with the known effects of low temperature on delaying phytochrome-far red absorbing form (Pfr) destruction, thus resulting in more Pfr being available to promote the responses. The difference between temperature treatments was greatest following a short light exposure, and much smaller following a long light exposure. When light was given as intermittent illumination the difference between subsequent temperature treatments was much less than when the same amount of light was given as continuous illumination.     

Red Light-Induced Shift of the Fluence-Response Curve for First Positive Curvature of Maize Coieoptiles

The fluence-response curve for first positive phototropic curvtureof dark-grown maize coleoptiles is shifted to ten-fold higherfluences if the coieoptiles are irradiated with red light 2h prior to the phototropic induction with blue light. Fluence-responsecurves for this red-induced shift were obtained with unilateralred irradiations 2 h prior to inductive blue pulses of differentfluences. They differ significantly depending on whether thered light was given from the same side as or the opposite sideto the respective inductive blue pulse, thus demonstrating thatthe red light effect is a local response of the coleoptile.The fluence-response curves for an inductive blue pulse in theascending part were compared with those for an inductive bluepulse in the descending part of the fluence-response curve forblue light induced phototropism. They are quite different inthreshold of red light sensitivity and shape for irradiationsfrom both the same and the opposite sides. This offers evidencefor the hypothesis that at least two different photosystemsare involved in phototropism, and that they are modulated differentlyby a red light preirradiation. All these fluence-response curvesindicate that it is possible to increase the response in thecoleoptile, if the red light preirradiation is given oppositeto the inductive blue pulse. This is supported by blue lightfluence-response curves obtained after a weak unilateral redpreirradiation. (Received September 11, 1986; Accepted October 18, 1986)     

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.     

Photoinduced Seed Germination of Oenothera biennis L: II. Analysis of the Photoinduction Period

The photoinduction period of Oenothera biennis L. seed germination was analyzed by varying the photoinduction temperature and by substituting red light pulses for continuous red light. At 24°C, seeds require 36 hours of continuous red light for maximal percent germination. The optimal photoinduction temperature is 32°C, with higher and lower temperatures being strongly inhibitory. A 30 minute exposure to far-red light, given immediately after a red light period of 1 to 36 hours, reduces germination by about 25%. Seeds escape from far-red inhibition with a half-time of 5 to 10 hours, depending on the length of the red exposure that precedes the far-red light. Periodic 15 minute pulses of red light can substitute for continuous red light in stimulating germination. Ted red light pulses, with 6 hours of darkness between successive pulses, cause maximal germination. The response to periodic red light is fully reversible by far-red light. Probit analysis of the periodic light response shows that as the length of the dark periods between successive pulses increases, less incident light is needed to induce germination but the population variance in light sensitivity remains constant. Probit analysis of the temperature response shows that as the photoinduction temperature increases from 16 to 32°C, less incident light is needed to induce germination and the population variance in light sensitivity also increases.     

Effect of continuous far red on betaxanthin and betacyanin synthesis

Seedlings of Celosia plumosa under prolonged irradiation with far red light synthesize chlorophyll α and betaxanthin. Levulinic acid and 2,4-dinitrophenol, inhibitors of chlorophyll synthesis and cyclic photophosphorylation respectively, reduce betaxanthin synthesis. Pigment formation is also inhibited by actinomycin-D and puromycin, but is unaffected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of noncyclic photophosphorylation. These findings are evidence of the involvement of photosynthesis through cyclic photophosphorylation, in the far red HER associated with betaxanthin synthesis. Under continuous far red seedlings of Amaranthus tricolor synthesize only chlorophyll α. Lack of betacyanin formation is ascribed to the inactive status of the genes involved in the pigment synthesis.     

Effects of Ca2+/Mg2+ chelators, SKF 525-A and rotenone on phytochrome-mediated betacyanin formation in Amaranthus seedlings

The effects of EDTA, EGTA, SKF 525-A (a selective inhibitor of cytochrome P-450) and rotenone were studied in betacyanin induction by 6 h red and 5 min far-red light, using etiolated, three-day-old Amaranthus caudatus L. half-seedlings. With 0.1 m M EDTA, EGTA and rotenone, and with 10 μ M SKF 525-A, mainly the far-red reversible betacyanin induction by red light was suppressed. Only in 0.1 m M rotenone was about 50% of that effect compensated by an increased far-red irreversible betacyanin induction. An unspecific inhibition was obtained with 0.1 m M SKF 525-A in both control and illuminated plants.
These results are consistent with the view that red light, but not far-red, causes Ca²⁺ efflux from both mitochondria and cytoplasm, whereas Ca²⁺ uptake is indicated mainly after illumination. The resulting switch in the coupling of the mitochondrial electron transport to a Ca²⁺ dependent one in cytochrome P-450 system via respiratory complex 1, appears to be responsible for the far-red reversibility. However, the bulk of the high irradiance reaction seems to be related to another secondary messenger, alternative to Ca²⁺.     

Evidence against an involvement of cyclic nucleotides in the induction of betacyanin synthesis by cytokinins.

1. A wide range of purine bases, nucleosides and cyclic nucleotides were shown to induce betacyanin synthesis in Amaranthus seedlings. 2. The induction of pigment by benzyladenine, dibutyryl cyclic AMP or cyclic AMP was not potentiated by aminophylline. Aminophylline was shown to inhibit Amaranthus cyclic AMP phosphodiesterase activity. 4. Incubation of seedlings with aminophylline inhibited the conversion of 6-[G--3H]benzyladenine into presumed 9- and 7-glucosylbenzyladenine. 5. Induction of betacyanin synthesis by 6-benzyladenine or by exposure to red light was not accompanied by changes in the total cyclic AMP content in seedlings. 6. It is concluded that the inducers tested act as cytokinin analogues; no evidence was obtained to support cyclic AMP as an intermediate in the induction process.     

Control of chlorophyll a synthesis by phytochrome and cryptochrome in the red alga Corallina elongata Ellis et Soland

Chlorophyll a synthesis in the red alga Corallina elongata is controlled by phytochrome and by a specific blue light photoreceptor. Although the estimated photoequilibrium of phytochrome is similar in blue and red light, the amount of chlorophyll accumulated is greater in blue light, which implies the action of cryptochrome, according to the criteria for the specific blue light photoreceptor involvement. The amount of chlorophyll synthesized is greater when the level of photoequilibrium approaches 65% (in blue and red light) than with higher levels (72.7% in white light and 70.8% in green light). The action of phytochrome is demonstrated by the induction of chlorophyll synthesis after red pulses and the reversion after far red pulses. The reversion is not complete but the percentage of reversibility is high (85-90%). The amount of chlorophyll accumulated is greater in darkness after the application of red light pulses than in white light after the same light pulses. The induction of chlorophyll synthesis is greater after red pulses than after continuous red light. The existence of a fast destruction of chlorophyll in continuous light is observed. This destruction is greater in the high photoequilibrium of phytochrome (70-72%). The turnover times and the induction mechanism of chlorophyll synthesis must be very fast. This indicates the existence of a possible rapid adaptation to the change in light quality and intensity in the marine system.     

Comparison of Three Phytochrome-mediated Processes in the Hypocotyl of Mustard

Anthocyanin synthesis, hair formation, and the synthesis of ascorbic acid oxidase are all phytochrome-mediated reactions occurring in the hypocotyl of mustard (Sinapis alba L.), controlled by phytochrome actually located in the hypocotyl. A comparison of these three reactions showed that in certain respects they differ greatly in their response to light. The ability of the seedling to respond to light by showing the three responses was strongly influenced by the state of development of the seedling. White light given very early after seed imbibition was unable to evoke any of the three reactions. By 50 hours after imbibition, all systems were fully inducible by light. The addition of actinomycin D to a fully competent seedling coincident with illumination strongly inhibited the development of all three responses. In contrast, the addition of cordycepin at this time inhibited the synthesis of anthocyanin and ascorbic acid oxidase but had no effect on hair formation. Cycloheximide inhibited all three responses when given up to several hours after light. This suggests the necessity for RNA and protein synthesis for light-induced expression of these reactions, and that the RNA species involved in the three reactions may have differing degrees of polyadenylation. The lag period between the onset of light and the first display of the response was 3 hours for anthocyanin and ascorbic acid oxidase synthesis, and about 5 hours for hair formation. Amounts of light sufficient to give large increases in the levels of ascorbic acid oxidase and hair formation gave a much smaller increase in anthocyanin synthesis. Hair formation and ascorbic acid oxidase synthesis showed a much greater sensitivity to induction at early stages of seedling development than did anthocyanin synthesis. Following an inductive light period, anthocyanin synthesis was sensitive to far red light inhibition for a period twice as long as the other two reactions. The differences in the response of the three reactions to light suggest that the phytochrome-mediated reactions which control their development also differ.     

Regulation of 5-Aminolevulinic Acid (ALA) Synthesis in Developing Chloroplasts : II. Regulation of ALA-Synthesizing Capacity by Phytochrome

When dark-grown cucumber (Cucumis sativus L.) seedlings previously exposed to white light for 20 hours were returned to darkness, the ability of isolated chloroplasts to synthesize 5-aminolevulinic acid dropped by approximately 70% within 1 hour. The seedlings were then exposed to light, and the synthetic ability of the isolated chloroplasts was determined. Restoration of the synthetic capacity was promoted by continuous white or red light of moderate intensity. Intermittent red light was also effective. Blue and far-red light did not restore the synthetic capability. Blue light given after a red pulse did not enhance the effect of the red light. Far-red light given immediately after each red pulse prevented the stimulation due to intermittent red light. Restoration of the biosynthetic activity by in vivo light treatments was inhibited by cycloheximide indicating the requirement for translation on 80 S ribosomes for the in vivo light response. These findings suggest that the majority of the plastidic 5-aminolevulinic acid synthesis is under phytochrome regulation.     

Intact plastids are required for nitrate- and light-induced accumulation of nitrate reductase activity and mRNA in squash cotyledons

Induction of nitrate reductase activity and mRNA by nitrate and light is prevented if chloroplasts are destroyed by photooxidation in norflurazon-treated squash (Cucurbita maxima L.) cotyledons. The enzyme activity and mRNA can be induced if norflurazon-treated squash seedlings are kept in low-intensity red light, which minimizes photodamage to the plastids. It is concluded that induction of nitrate reductase activity and nitrate reductase mRNA requires intact plastids. If squash seedlings grown in low-intensity red light are transferred to photooxidative white light, nitrate reductase activity accumulates during the first 12 hours after the shift and declines thereafter. Thus photodamage to the plastids and the disappearance of nitrate reductase activity and mRNA are events separable in time, and disappearance of the enzyme activity is a consequence of the damage to the plastids.     

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.     

Light-induced Adhesion of Spirogyra Cells to Glass

Adhesion of Spirogyra (tentatively, Spirogyra fluviatilis) cells to glass is described. The cells of an algal filament can adhere to a substrate only when they are located at the end of the filament. Rapid adhesion is induced by blue-violet light (blue adhesion) as well as by temperature shift (about 6 C → about 22 C) or shaking (dark adhesion). Adherent cells detach in 1 hour in the absence of one of these stimuli. Slow adhesion is induced by red light (red adhesion) 1 hour after irradiation, and may be controlled by phytochrome. A cell once caused to adhere by red light does not release from the glass.     

Interactions of light and a temperature shift on seed germination

Germination of Rumex obtusifolius L. seeds is potentiated to an observable degree in 2 minutes by a single shift in temperature from 20 to 35 C. Half-maximal potentiation requires less than 32 minutes at the higher temperature. Similar sensitivities to shifts in temperature were observed for seeds of Barbarea vulgaris, R.Br. B. verna (Mill.) Asch., and Lepidium virginicum L. A shift in temperature interacts strongly with change in form of phytochrome induced by light on germination of the four kinds of seeds. The potentiated effects for R. obtusifolius are only moderately affected by 40 μm cycloheximide. Both the temperature shift and light actions are apparently independent of processes of synthesis necessary for growth.