Significance of Cyclic Nucleotides in Amaranthin Synthesis by Amaranthus caudatus Seedlings

Explants of 72–76 h old Amaranthus caudatus seedlingssynthesize the betalain pigment amaranthin in response to light.Light can be replaced with a cytokinin or a cyclic nucleotidewith an N⁶-substituent. Cyclic 3'5'-AMP shows only weak activityand that only at high unphysiological concentrations. Even cyclic2'3'-AMP, which docs not act as a ‘second messenger’,induces amaranthin synthesis to a greater degree than cyclic3',5'-AMP. But N⁶-monobutyryl-cyclic 3',5'-AMP and N⁶-2'-O-dibutyryl-cyclicAMPshow high activity, higher even than kinetin at its optimumconcentration of 10^–5 M. 2'-O-Monobutyryl-cyclicAMP, onthe other hand, is considerably less active, suggesting thatN⁶-substitution of the adenine ring is responsible for the enhancedactivity. N⁶-Propionyl, butyryl and valeryladenines are allhighly active, indicating that the cyclic monophosphate moietyis unnecessary for this response. All the compounds tested,including cyclic 3',5'-AMP, show additive effects, but thereis no amplification of the response, typical of second messengeraction. Inhibition of amaranthin synthesis imposed by hadacidin, isrelieved by kinetin, DBc AMP, N⁶-monobutyryl-cAMP and N⁶-butyryladenine. Cyclic 3',5'-AMP is weakly active in this regard. Asnatural cytokinins are N⁶-substituted adenine compounds, andas only N⁶-substituted cyclic nucleotides are able to mimicthe effect of cytokinin, it is concluded that these cyclic nucleotidesfunction as cytokinin analogues and not as ‘second messengers’'. Amaranthus caudatus, amaranthin, cytokinins, cyclic nucleotides     

Cyclic Nucleotides and In Vitro Plant Cultures: I. INDUCTION OF ORGANOGENESIS IN TOBACCO (NICOTIANA TABACUM CALLUS CULTURES

Mangat, B. S. and Janjua, S. 1987. Cyclic nucleotides and invitro plant cultures. I. Induction of organogenesis in tobacco(Nicotiana tabacum) callus cultures.—J. exp. Bot. 38:2059–2067. The possibility that cyclic nucleotides have a mediatory rolesimilar to cytokinins in plant tissue cultures was examined.Calli obtained from tobacco pith tissue were incubated on growthmedia supplemented with either cyclic AMP, cyclic GMP, adenosineor guanosine, in concentrations ranging from (mg dm^–3)0 to 2·0 together with 2·0 mg dm^–3 of IAA.Results were compared with identical calli grown on media containingcomparable amounts of kinetin and IAA. Increase in callus growthwas observed on all media containing cyclic AMP, cyclic GMP,adenosine, guanosine or kinetin. Adenosine or guanosine didnot promote organogenesis. Low concentrations (0·02 and0·05 mg dm^–3) of kinetin stimulated extensive rootdevelopment. Some root formation was also elicited with higheramounts of cyclic AMP (0·1 and 0·2 mg dm^–3)or cyclic GMP (0·2 and 0·5 mg dm^–3). Bothkinetin and cyclic GMP promoted shoot differentiation. However,in contrast to kinetin, cyclic GMP induced organogenesis atlower concentrations (0·02 and 0·1 mg dm^–3).The addition of 2·0 mg dm ^–3 of cyclic AM P toIAA-free growth media elicited shoot differentiation. This wasalso the case with a similar concentration of kinetin or cyclicGMP. Results suggest cytokinin activity for the two cyclic nucleotides. Key words: Tobacco, Nicotiana tabacum, tissue culture, cyclic nucleotides, cyclic AMP, cyclic GMP organogenesis     

Significance of Spermidine in the Initiation of Adventitious Buds in Stem Segments of Torenia

When stem segments of Torenia fournieri Lind. were culturedin vitro, the initiation of adventitious buds, which is usuallyinduced by cytokinin, was induced by application of polyamines,such as putrescine and spermidine. Addition of arginine hada slight inductive effect. When cyclohexylamine, an inhibitorof spermidine synthase, was added simultaneously with putrescine,induction of the initiation of buds by putrescine was stronglyinhibited. However, application of the inhibitor together withspermidine had no effect on the spermidine-induced initiationof buds. The induction of initiation of buds by a cytokinin,by a calcium ionophore, by cyclic AMP, and by a phorbol ester,which was accompanied in each case by elevation of the levelsof endogenous spermidine, was also inhibited by cyclohexylamine.These results suggest the involvement of spermidine in the initiationof adventitious buds in stem segments of Torenia. ²Present address: Radiation Effects Research Foundation, Hijiyamakouen5-2, Minami-ku, Hiroshima, 732 Japan.     

Phototropic sensitivity in Vaucheria geminata regulated by 3',5'-cyclic AMP

Exogenous 3',5'-cyclic AMP in physiological concentrations inhibitsthe phototropic bending of Vaucheria geminata without affectinggrowth. Related substances known to change the intracellularcyclic AMP level also alter phototropic sensitivity. This indicatesthat the steering process (i.e., initial phototropic response)is separate from growth itself. Cyclic AMP extracted from this alga amounted to 0.2–0.3nmole per g fresh weight, or about 3 µM on a cytoplasmvolume basis. This value coincides with expected results fromexperiments with exogenous cyclic AMP. The results suggest thatendogenous cyclic AMP may play an essential role in the gain-controllingprocess of phototropism. ¹ Present address: Institute for Agricultural Research, TohokuUniversity, Sendai 980, Japan. (Received November 26, 1976; )     

The possible role of cyclic AMP in the control of genetic tumor induction

Young seedlings of the tumor-prone amphiploid Nicotiana suaveolensx N. langsdorffii, grown aseptically on nutrient medium, weretreated with 1x10^–2 M cyclic adenosine 3': 5'-monophosphate(cyclic AMP). The incidence of tumor formation was scored atregular intervals subsequent to exposure. Cyclic AMP causeda significant reduction in the rate of tumor formation. In addition,untreated plants grown on nutrient medium were harvested atregular intervals after the seeds had been sown. Cyclic AMPwas extracted, partially purified, and assayed radioimmunologically.The endogenous level of cyclic AMP in stem tissue was highestin young seedlings, rapidly fell reaching a low point in 18day-old plants, and eventually leveled off. The presence ofindoleacetic acid (IAA) in the growth medium at a final concentrationof 2x10^–5 M prevented the decline in cyclic AMP that occurredin seedlings grown on unsupplemented medium. (Received May 21, 1976; )     

Effect of Growth Regulators and Light on Pollen Germination and Pollen Tube Growth in Pinus roxburghii Sarg

Pine (Pinus roxburghii) pollen grown in suspension cultureswas used to study the effects of growth regulators and lightconditions on germination and pollen tube growth. Indol-3-ylacetic acid, gibberellic acid, ethylene, abscisic acid and cyclicAMP (cAMP) at low concentrations (1–10 mg 1^–1) promotedgermination and tube growth. Addition of 1 and 10 mg 1^–1cAMP to any of the growth regulators had a promotory effect.Pollen tube growth decreased in white light as compared to thedark, and was increased in red light. Far-red light counteractedthe effect of red light. The effect of growth regulators incausing the enhanced tube growth appears to be manifested throughsubstances such as cAMP, and phytochrome seems to be involved. Pinus roxburghii, pine, pollen germination, pollen tube growth, growth regulators, cyclic AMP, phytochrome     

Removal by chemicals of photoperiodic light requirements of Lemna gibba G3

Both the initial and the terminal 1 hr portions of the subjectiveday fraction, namely the L1- arid L2-phases, of a 24 hr daymust be illuminated in order for the day to be perceived asa long day in the min-LD determination by the long-day plant,Lemna gibba G3 (9). The light requirement of the L1-phase wassatisfied by a 10 min red light pulse given at the beginningof the phase. The red light effect was erased by a subsequent10 min far-red light, indicating phytochrome-mediated processesoccurring in the L1-phase. The light requirement of the L2-phasewas satisfied by blue or far-red light given during the terminal10 min period of the phase; there was no indication of phytochromeinvolvement. The light action on the L1-phase was replaced by10^–5 M of cyclic AMP or 10^–7 M of DL-isoproterenol.The isoproterenol action was antagonized by 10^–7 M ofDL-propranolol. Cyclic AMP (10^–5 M) combined with salicylicacid (10^–6 M), which can remove the light requirementof the L2-phase (10), rendered a completely dark day physiologicallyequivalent to a long day. Acetylcholine (10^–5 M) exertednyctomimetic action on the L1-phase of the second light day.The action of acetylcholine was antagonized by cyclic AMP (10^–5M). The L2-phase required no light in the presence of 10^–7M of DL-propranolol, and this propranolol action was not affectedby isoproterenol. These findings suggest changes in membranepermeability caused by the light given during the L1- and L2-phases. (Received July 7, 1976; )     

Involvement of Cyclic AMP in Adventitious Bud Initiation of Torenia Stem Segments

Adventitous bud initiation in epidermis of Torenia stem segmentscultured in vitro, which is usually induced by cytokinin, couldbe induced by application of dibutyryl cyclic AMP in the absenceof cytokinin. Similar stimulatory effects on bud initiationwere observed when various promoting reagents to accumulateendogenous cyclic AMP were added, such as activators for adenylatecyclase, forskolin and prostaglandin E₁, and inhibitors of cyclicnucleotide phospho-diesterase, theophylline and isobutyl methylxanthine. Endogenous content of cyclic AMP in Torenia stem segmentswere increased by the application of the above chemicals, calciumionophore or cytokinin. These results suggested that endogenousconcentrations of cyclic AMP were involved in adventitious budinitiation of Torenia stem segments. Furthermore, some inhibitorsof protein kinases inhibited bud initiation induced by cyclicAMP-accumulating reagents. (Received August 15, 1989; Accepted November 4, 1989)     

Cytokinin-Mediated Translational Control of Protein Synthesis in Cultured Cells of Glycine max

Polyribosome formation was stimulated by cytokinin treatmentof cultured cells of Glycine max cv. Funk Delicious. When suspensioncultures were given 0·5 µM zeatin after 24 h inculture in medium lacking a cytokinin, a nearly 2-fold increasein the polyribosome/monoribosome ratio occurred over the subsequent3 h. The effect of actinomycin D and of 5-fluorouridine on RNAsynthesis and on the polyribosome/monoribosome ratios of thesecells was examined. Actinomycin D at 5 and 20 µg/ml^–1inhibitedtotal RNA synthesis by 39 and 60%, respectively, as measuredby [³H]uridine incorporation into acid-precipitable material.The degree of inhibition of precursor incorporation into polyribosomalRNA was similar. At 0·1 mM, 5-fluorouridine inhibited[³H]uridine incorporation by 76%, and [³H]guanosine incorporationby 66% into polyribosomal RNA after 3 h of treatment. Fractionationof the polyribosomal RNA by oligo(dT)-cellulose chromatographydemonstrated that low concentrations of both actinomycin D (5µg ml^–1) and 5-fluorouridine (0·1 mM) inhibitedthe synthesis of ribosomal RNA to a greater extent than thepoly(A)-containing fraction of the messenger RNA. Synthesisof the poly(A)-containing RNA was inhibited by 24% with 5µgml^–1 actinomycin D and by 30% with 0·1 mM 5-fluorouridine.At the above concentrations, these two inhibitors reduced thepolyribosome/monoribosome ratio of the cytokinin-deprived cellsover a 3 h period, but they did not prevent cytokinin-inducedpolyribosome formation. These results provide further evidencethat cytokinin regulates polyribosome levels through an effecton protein synthesis at the translational level     

Transport and Metabolism of Dihydrozeatin Riboside in Germinating Lupin Seeds

[³H]-dihydrozeatin riboside was applied selectively to the embryonicaxes or to the cotyledons of germinating lupin (Lupinus luteusL. cv. Weiko III) seeds 6 h following the start of imbibition.There was little transport of dihydrozeatin riboside from embryoto cotyledons up to 6 h after the application, but a substantialamount of radioactivity had moved into the cotyledons at theend of the 10 h incubation period. However, there was no detectablemovement of [³H]-dihydrozeatin riboside from the cotyledonsto the embryonic axis. This indicated a highly polarized movementof cytokinins during the early stages of seed germination. Exogenouslyapplied [³H]-dihydrozeatin riboside was found to be very stable,both when applied to the embryonic axes and cotyledons of intactseed, or following excision, and there was little metabolismwith only small amounts of radioactivity found associated withdegradative metabolites. The embryonic axis of this specieshas recently been found to synthesize cytokinins within 12 hfrom the start of imbibition, and the results of this studyindicate that the embryo-derived cytokinin is probably transportedto the cotyledons where it accumulates and subsequently participatesin the control of cotyledon function. Key words: Lupinus luteus, cytokinin transport and metabolism, dihydrozeatin riboside, seed germination     

Role of Cytokinin in Vessel Regeneration in Wounded Coleus Internodes

Cytokinin was found to be a controlling or limiting factor invessel regeneration around a wound in internodes of Coleus blumeiBenth. in which the endogenous cytokinin level was minimized.The cytokinin was applied in aqueous solution to the base ofexcised, mature internodes that had an active vascular cambium.Each internode also received IAA in lanolin at its apical end.Under low (0.1 %, w/w) or high (10%, w/w) auxin concentrations,the control internodes (without exogenous cytokinin) exhibitedsmall amounts of vessel regeneration. At appropriate concentrationszeatin, kinetin and 6-benzylamino-purine (BAP) induced a significantincrease in vessel regeneration around the wound. The threecytokinins also induced novel patterns of supplementary regenerationfurther from the wound surface. Kinetin and BAP showed the strongestpromoting effect at 5 and 10 µg ml^–1, while zeatinwas most effective at 20 µg ml^–1. At a low (0.1%) auxin level zeatin was the most effective cytokinin, whereaskinetin was the most effective one at high (1 %) auxin. An inhibitoryeffect on vessel regeneration was observed at the highest kinetinconcentration tested (50 µg ml^–1). The regenerationof vessels induced by cytokinin was very polar. Many more regeneratedvessel members differentiated below the wound than above it,and the regeneration process proceeded acropetally from thebase of the internode to its upper parts. Our results implya basipetal polar increase in cambium responsiveness along thestem axis from internode 5 to 7. The possible significance ofsuch a basipetal increase in cambium sensitivity in wood formationin trees is discussed. Auxin, Coleus blumei, cytokinin, vascular differentiation, vessel regeneration, wound xylem     

Formation of guanosine 3′,5′-cyclic monophosphate by thyroliberlin in cultured rat pituitary cells a possible role in stimulation of prolactin synthesis

In a clonal strain of rat pituitary tumour cells (GH₄C₁ cells), thyroliberin stimulated prolactin secretion and synthesis: effects that could be demonstrated after 5 min and 4–5 h of treatment, respectively. Within 0.5–5 min after addition of thyroliberin, maximal increases (2–4 hold) in cellular cyclic GMP concentrations were observed, and this rise preceded or occurred simultaneously with that of cyclic AMP. After 60 min of treatment the concentrations of the cyclic nucleotides had returned to control values. Half maximal and maximal stimulation of cyclic GMP elevations were obtained with approx. 2·10⁹ and approx. 27·10^?9 thyroliberin, respectively. Aminophylline increased both cyclic GMP and cyclic AMP, and potentiated the stimulatory effects of thyroliberin on both cyclic nucleotides. The dibutyryl derivative of cyclic GMP (10^?4–10^?6 M) stimulated prolactin synthesis, but not hormone release. Prostaglandin E₂ (3·10^?7 M) stimulated cellular cyclic AMP concentrations, but did not affect cyclic GMP levels. We conclude that thyroliberin in the GH₄C₁ ccell strain stimulates cyclic GMP formation, in addition to elevate cyclic AMP concentrations. The stimulatory effect on cyclic GMP is probably not secondary to the rise in cyclic AMP concentration, since prostaglandin E₂ elevates only cyclic GMP is involved in the action of thyroliberin on prolactin, the present results suggest a role on hormone synthesis.     

Inhibition of Cell Division and DNA Synthesis by Gibberellin in Isolated Zinnia Mesophyll Cells

A culture system of isolated mesophyll cells of Zinnia eleganswas used to examine the action of gibberellic acid (GA) on celldivision. Isolated Zinnia mesophyll cells cultured in a mediumcontaining auxin and cytokinin reinitiated cell division ina partly synchronized manner. When mesophyll cells isolatedfrom 21-day-old seedlings were used, GA added to the culturemedium at concentrations of 1 x 10^–6 M or higher suppressedthe initial rise in the number of divided cells. Tracer experimentswith [³H]-dThd revealed that GA treatment inhibited the incorporationof [³H]-dThd into DNA in the nucleus without inhibiting theuptake of [³H]-dThd into the cells, indicating that GA inhibitedDNA synthesis. GA applied at 48 h inhibited the incorporationof [³H]-dThd into DNA during the following 24 h, but GA appliedat 72 h did not inhibit the incorporation during the subsequent24 h. This suggests that GA affects the process of reinitiationof DNA synthesis, but does not affect DNA synthesis once cellshave become proliferative. (Received January 14, 1986; Accepted March 31, 1986)     

Relative Importance of Ion Exclusion, Secretion and Accumulation in Spartina alterniflora Loisel.

The extent to which Spartina alterniflora Loisel. excluded,secreted or accumulated the major seawater ions (Cl^-, SO^2-₄,Na⁺, K⁺, Mg²⁺, and Ca²⁺) was investigated under varying salinitytreatments. From a quantitative viewpoint, ion exclusion wasmost prominent and accounted for 91–97% of the theoreticalmaximum ion uptake as a result of transpiration and growth.Of those ions taken up, approximately half was secreted fromthe shoots. Relative to K⁺, a disproportionate amount of Na⁺was excluded at the roots and secreted by the shoots. The concentrationwithin the tissues of S. alterniflora did not change with salinitytreatment for the majority of the ions examined, but Na⁺ wasmore than twice as concentrated at 40 g dm^-3 than at lOgdm^-3.Calculations of the flux of ions from salt marsh sediments tothe flood water via shoot secretion or stem/leaf turnover indicatethat these processes may be important to the ecology of S. alternifloraas mechanisms that limit the accumulation of salt within theroot zone.     

Stimulation of gibberellin-induced germination in lettuce seeds by cyclic 3',5'-adenosine monophosphate

Cyclic 3',5'-adenosine monophosphate and sodium dibutyryl cyclic3',5'-adenosine monophosphate had no effect on dark-germinationof light-sensitive lettuce {Lacluca sativa L., var. Grand Rapids)seeds when given alone. Cyclic 3',5'-adenosine monophosphate,however, synergistically enhanced the 3 µ 10^–5 Mgibberellic acid-induced germination, but showed no such effecton kinetin-stimulated germination. 5'-AMP, in contrast to cyclic3',5'-adenosine monophosphate, had no effect on gibberellicacidinduced germination. (Received August 8, 1971; )     

Effects of Cyclic AMP on the Activity of Soluble Protein Kinases in Lemna paucicostata

Three protein kinases which phosphorylate histone were isolatedfrom cellular extract of Lemna plants. They were separated byelution from DEAE-Sephacel column and referred to as PI, PITand PHI. The PI protein kinase activity was partially inhibitedby 10µM cyclic AMP, cyclic GMP or cyclic IMP, while thePII enzyme was activated in the presence of these cyclic nucleotides.The PIII enzyme was cAMPindependent, but slightly inhibitedby cyclic CMP and cyclic UMP. The molecular weights of thesethree protein kinases were 165,000, 85,000 and 145,000, respectively,as estimated from Sephacryl S-300 gel filtration. A single cyclicAMP-binding protein was detected in the PII enzyme fractionby using the photoaffinity cAMP-analogue, 8-N₃-cAMP. The proteinwhich specifically bound [³H]-8-N₃-cAMP had an apparent molecularweight of 48,000 as determined by SDS-polyacrylamide gel electrophoresis.The phosphorylation of cellular proteins in Lemna was examinedby SDS-polyacrylamide gel electrophoresis. Four phosphorylatedpolypeptides were detected, the phosphorylations of which werestimulated by cAMP. The molecular weights of these four polypeptideswere 59,000, 19,000, 16,000 and 14,000, respectively. (Received January 26, 1983; Accepted April 13, 1983)     

Reversal of sucrose inhibition of Lemna flowering by adenine derivatives

Sucrose inhibition of flowering in Lemna perpusilla 6746 wasat least partially reversed by 5'-AMP, cyclic 3',5'-AMP, 5'-ADP,5'-ATP and K₂HPO₄. These results are in contrast to those reportedfor L. gibba in which reversal was effected by cyclic AMP, butnot by other adenine derivatives. ¹ This work was supported by National Science Foundation GrantGB-12955. (Received June 11, 1973; )     

Swelling of Etiolated Oat Protoplasts Induced by cAMP and Red Light

Etiolated oat protoplasts were treated with dibutyryl cAMP tostudy possible function of cAMP in the development by measuringthe protoplast swelling. The mean diameter of protoplasts inthe absence of any chemical treatment was 33.58±1.26(SE) µm, which increased to 36.96±0.86 µmin the presence of 100 µM dibutyryl cAMP. Prostacyclin,a potent activator of adenyl cyclase, also showed a significantswelling effect (diameter 38.01±0.98 µm). Red lightalso elicited the swelling of protoplasts (40.26±0.8µm). ¹Present address: Department of Biology, Pusan National University,Pusan 607, Korea. ²Present address: Department of Horticulture, Cheju NationalUniversity, Cheju 590, Korea. ³Present address: Department of Biological Sciences, Texas TechUniversity, Lubbock, TX 79409, U.S.A. (Received June 29, 1985; Accepted November 18, 1985)     

Cytokinin Regulation of Flower and Pod Set in Soybeans (Glycine max(L.) Merr.)

Exogenous application of cytokinin to raceme tissues of soybean(Glycine max(L.) Merr.) has been shown to stimulate flower productionand to prevent flower abortion. The effects of these hormoneapplications have been ascertained for treated tissues, butthe effects of cytokinins on total seed yields in treated plantshave not been evaluated. Our objectives were to examine theeffects of systemic cytokinin applications on soybean yieldsusing an experimental line of soybeans, SD-87001, that has beenshown to be highly sensitive to exogenous cytokinin application.Soybeans were grown hydroponically or in pots in the greenhouse,and 6-benzylaminopurine (BA) was introduced into the xylem streamthrough a cotton wick for 2 weeks during anthesis. After theplants had matured, the number of pods, seeds per pod, and thetotal seed weight per plant were measured. In the greenhouse,application of 3.4 x 10^-7 moles of BA resulted in a 79% increasein seed yield compared with controls. Results of field trialsshowed much greater variability within treatments, with consistent,but non-significant increases in seed number and total yieldsof about 3%. Data suggest that cytokinin levels play a significantrole in determining total yield in soybeans, and that increasingcytokinin concentrations in certain environments may resultin increased total seed production. Copyright 2001 Annals ofBotany Company Glycine max, soybean, flower abortion, cytokinin, 6-benzylaminopurine, hydroponic, seed yield, wicking