The influence of light on the cytokinin content ofAmaranthus seedlings

The influence of light, particularly blue and red light, on the cytokinin content of seedlings ofAmaranthus caudatus was studied. Cytokinin content was determined by two different bio-assays (amaranthin accumulation byAmaranthus seedlings and mtrate-reductase activity ofAgrostemma embryos). In both bio-assays similar results were obtained. Oytokinin content is increased, especially by blue light. It is suggested that especially blue light promotes amaranthin accumulation inAmaranthus seedlingsvia the increase of cytokinin content of tissues. The results support our hypothesis on cytokinin action.     

The hormonal control of betacyanin synthesis in Amaranthus caudatus

Gibberellic acid (GA₃) inhibits amaranthin synthesis whereas the growth retardant, phosphon D, enhances pigment levels in A. caudatus seedlings exposed to light. No effect was observed on chlorophyll and carotenoid synthesis. Radioactive tyrosine and DOPA were incorporated into amaranthin. The specific activity of amaranthin synthesised in the presence of ¹⁴C-tyrosine or ¹⁴C-DOPA in seedlings treated with GA₃ is higher than water controls. The specific activity of pigment from phosphon D treated tissue is relatively low. GA₃ treated tissue has lower active tyrosine and DOPA pools compared to phosphon treated seedlings. Tyrosine and DOPA-oxidase activity increases in GA₃ treated and H₂O control seedlings exposed to light. Kinetin stimulates the synthesis of amaranthin in dark-grown seedlings and this is not overcome by simultaneous GA₃ application. Dark-grown seedlings treated with different kinetin concentrations and incubated in ¹⁴C-tyrosine synthesise radioactive amaranthin of similar specific activity. Kinetin treatment of dark-grown seedlings brings about an increased tyrosine and DOPA-oxidase activity. The results indicate that GA₃ controls the production and/or availability of tyrosine whereas kinetin can mimic light treatment and controls the utilisation of tyrosine probably by bringing about the synthesis or activation of tyrosine and DOPA-oxidase protein.     

Effect of a cytokinin antagonist on cytokinin and light-dependent amaranthin synthesis inAmaranthus Tricolor seedlings

InAmaranthus tricolor seedlings, amaranthin synthesis can be induced under the effect either of a cytokinin or of white light. The 3-methyl-7-(n-pentylamino)pyrazolo(4,3-d)pyrimidine (PAMPP), a cytokinin analog that strongly inhibits the growth of tobacco callus, antagonizes the stimulating effect of cytokinin as well as stimulation by light. In dose-response terms, the inhibitory effect of PAMPP was described as competitive with respect to N⁶-benzyladenine (b⁶Ade) or light. The inhibition by PAMPP of the b⁶Ade amaranthin test response or the inhibition by this cytokinin analog of the light amaranthin test response were both reversed by either subsequent light or b⁶Ade treatment.     

Effects of light and kinetin on amaranthin synthesis induced by cAMP

The effects of cyclic 3′,5′-adenosine monophosphate (cAMP) on amaranthin synthesis in the dark, or in the presence of kinetin or light were investigated in isolated cotyledons of Amaranthus tricolor and A. caudatus. The results suggest that sites or modes of action of cAMP and kinetin are not separated and differ from those of light and that the nucleotide cannot be considered a messenger involved in amaranthin formation stimulated by kinetin or by light.     

Biosynthesis of amaranthin in Celosia plumosa

Seedlings of a yellow betaxanthin-producing variety of Celosia plumosa when fed with appropriate precursors are capable of synthesizing the red-violet pigment normally present in red varieties of the same species, namely amaranthin. Synthesis of amaranthin occurs in seedlings following administration of betanidin and betanin but much greater accumulation was observed after feeding cycloDOPA and its 5-O-β-d-glucoside. Possible pathways in the biosynthesis of amaranthin are discussed.     

Influence of calcium and calcium and calmodulin antagonists on the cytokinin-induced amaranthin accumulation inAmaranthus tricolor

The concentration of kinetin and kinetinriboside plays an essential role in the induction of amaranthin accumulation in cotyledons ofAmaranthus tricolor during germination. The dose/effect ratio shows that kinetin induced 3- to 3.5-fold more amaranthin than kinetinriboside at the same molecular concentration. Various concentrations of exogenous Ca²⁺ did not influence the effects of kinetin on the betacyanin synthesis. However, when Ca²⁺ was applied together with kinetinriboside, the amaranthin production was stimulated. Time-course experiments show a lag phase of 16 h starting from the incubation with kinetin and a distinct increase of amaranthin thereafter. If the seedlings were treated simultaneously with kinetin and Ca²⁺, the increase of amaranthin started after 12 h. At 16 h of incubation in kinetin/Ca²⁺, the amount of amaranthin increased significantly compared to controls incubated with kinetin alone. If Ca²⁺ ions (16 h kinetin/Ca²⁺ incubation) were removed from the medium after 2 h, 4 h, and up to 14 h, the amaranthin content was enhanced compared to controls without Ca²⁺. The stimulating effect was highest in the presence of Ca²⁺ for 8 h. These data show that exogenous Ca²⁺ stimulated the amaranthin synthesis mainly during the first 12 h of incubation. The Ca²⁺ antagonists EGTA, chlorotetracycline, and CoCl₂ reduced the amaranthin content up to 80%. The calmodulin antagonists chloropromazine and trifluoperazine inhibited the betacyanin accumulation up to 97% when applied at the beginning of the incubation. Neither Co²⁺ nor trifluoperazine after 12 h of preincubation in kinetin had inhibiting effects on the amaranthin production. Therefore, we presume that a specific period of competence is required for calmodulin-mediated Ca²⁺ effects on the accumulation of amaranthin induced by cytokinins in the seedlings ofAmaranthus tricolor.     

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     

Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1

Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation.     

Light control of amaranthin synthesis in isolated Amaranthus cotyledons

The effect of light on the amaranthin synthesis stimulated by exogenous precursors has been studied in isolated cotyledons of Amaranthus tricolor and A. caudatus. The results indicate that light acts at the level of the formation of the dihydropyridine moiety of the pigment.     

Effect of a cytokinin antagonist on cytokinin and light-dependent amaranthin synthesis inAmaranthus Tricolor seedlings

InAmaranthus tricolor seedlings, amaranthin synthesis can be induced under the effect either of a cytokinin or of white light. The 3-methyl-7-(n-pentylamino)pyrazolo(4,3-d)pyrimidine (PAMPP), a cytokinin analog that strongly inhibits the growth of tobacco callus, antagonizes the stimulating effect of cytokinin as well as stimulation by light. In dose-response terms, the inhibitory effect of PAMPP was described as competitive with respect to N⁶-benzyladenine (b⁶Ade) or light. The inhibition by PAMPP of the b⁶Ade amaranthin test response or the inhibition by this cytokinin analog of the light amaranthin test response were both reversed by either subsequent light or b⁶Ade treatment.     

In vitro morphogenic characteristics of phytochrome mutants in Nicotiana plumbaginifolia are modified and correlated to high indole-3-acetic acid levels

The involvement of indole-3-acetic acid (IAA) in the integration of the light signal perceived by phytochrome during the morphogenesis of plants was investigated in Nicotiana plumbaginifolia Viviani. The chromophore mutant pew1, deficient in all the phytochrome types, and the aurea-like mutant pew2, which appears to be specifically deficient in phytochromes expressed in darkness, were analysed for IAA-related morphogenic effects such as rooting, shooting and callus formation. We observed, in the absence of exogenously applied hormones, abundant root formation by the pew2 mutant. The pew1 mutant exhibited callus formation in the presence of gibberellins and cytokinins when the wild type did not. The previously described lethality of the double mutant pew1–pew2 was shown to be hormone-dependent since, in the light, exogenously applied auxin and cytokinin (0.1 mg·1?1 each) led to plant regeneration from calli and subsequent normal development. These observations suggested an increase in the auxin/cytokinin ratio as a consequence of the phytochrome mutations. We correlated these morphogenic characteristics with high IAA levels in the mutants. The difference in IAA accumulation in the two mutants indicates that among the different phytochromes expressed by N. plumbaginifolia, the light-expressed isoforms play a major role in the control of IAA levels.     

Effects of light and growth regulators on adventitious bud formation in horseradish (Armoracia rusticana)

Summary To clarify that the presence of Ri T-DNA genes are not prerequisite for the light-induced bud formation in horseradish (Armoracia rusticana) hairy roots, leaf and root segments of nontransformed horseradish plants were used as explants. Bud formation from nontransformed tissues was observed in hormone-free medium under 16 h daylight conditions, but not under continuous darkness. To investigate the effects of growth regulators on bud formation, leaf and root explants were treated with auxin (1-naphthaleneacetic acid; NAA) and / or cytokinin (6-benzyl-aminopurine; BA). The most effective treatment in the dark to stimulate bud formation was BA at 1 mg·1^-1. These results show that adventitious bud formation in horseradish can be induced by light and growth regulators, and especially cytokinin, may be involved in bud formation, irrespective of whether the tissues were transformed with Ri T-DNA.Abbreviations BA 6-benzyl-aminopurine - NAA 1-Naphthaleneacetic acid - MS Murashige & Skoog (1962) medium     

Nitrogen fertilizer increases spikelet number per panicle by enhancing cytokinin synthesis in rice

Key message

Nitrogen fertilizer enhances local cytokinin synthesis to increase flower numbers in the panicles of rice. Localized cytokinin biosynthesis is an important response to nitrogen.

Abstract

Flower number per panicle is one of the most important traits in rice productivity determination. The number of flowers is established in the early stages of panicle development. Nitrogen fertilizer application before panicle initiation is well known to increase flower number. Nitrogen increases cytokinin (CKs) biosynthesis in plants, and CKs have very similar effects as nitrogen fertilizer on panicle branching. The effects of nitrogen fertilizer on panicle branching may be mediated by CKs, in which accumulation in the inflorescence meristem can regulate panicle development, resulting in increased numbers of flowers and branches. Adenosine phosphate-isopentenyltransferase (IPT) catalyzes the rate-limiting step of CKs biosynthesis. We analyzed the effect of nitrogen fertilizer (urea) on the expression of OsIPT genes (OsIPTs). The results showed that OsIPTs were markedly increased, and CKs accumulated in panicle when nitrogen fertilizer was applied. CKs biosynthesis in the roots and leaves was not up-regulated by nitrogen. These results suggest that nitrogen fertilizer enhances local CKs synthesis to increase flower numbers in the panicles of rice. Localized CKs biosynthesis is an important response to nitrogen.     

Use of 5,6-dichloro-l-β-D-ribofuranosylbenzimidazole to distinguish light stimulation from cytokinin stimulation of amaranthin synthesis in Amaranthus tricolor

In Amaranthus tricolor seedlings, amaranthin synthesis can be induced by cytokinins and (or) by light. It was found that 5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB) was a specific inhibitor of the cytokinin response whereas the light response was unchanged. DRB also inhibited the negative response of pigment production to an anticytokinin, 7-(pentylamino)-3-methylpyrazolo(4,3-d) pyrimidine (PAMPP).DRB can be considered as a specific inhibitor of the physiological responses to cytokinins. The evolution of the inhibition percentage versus cytokinin or anticytokinin concentration shows that DRB is not a `competitor' of cytokinins.The experiments suggest that cytokinins or anticytokinins act as modulators of a fundamental light activated reaction of amaranthin synthesis.Address where proofs should be sent: Laboratoire de Physiologie végétale, Faculté des Sciences et des Techniques, Université de Nice, F-06034 Nice Cedex, France     

Morphactin-induced changes in the cytokinin effect on tissue and organ cultures ofNicotiana tabacum

Summary The influence of a morphactin, chlorflurenol-methylester (CFM), on the growth, the morphogenesis and the isoelectric peroxidase pattern was investigated in both callus cultures (two different tissue culture strains) and multiple bud cultures ofNicotiana tabacum var.Wisconsin. CFM (range of concentration between 10^–6g/ml and 10^–4g/ml) was applied singly, or in combination with a cytokinin, benzylaminopurine (BAP), or with an auxin, indoleacetic acid (IAA), or with IAA plus BAP.In general, the callus growth was inhibited under the influence of CFM. In some of the experiments carried out in hormone-free media, growth stimulation was observed. Even minimal inhibition or stimulation of the callus growth was always accompanied by characteristic changes in the peroxidase patterns.The following results show the influence of the morphactin CFM on cytokinin effects (endogenous cytokinin or equally the exogenously applied cytokinin, BAP). (1) In the multiple bud cultures, BAP and CFM (both substances combined with IAA) similarly caused inhibition of root formation and stimulation of bud formation. The bands in the peroxidase patterns, characteristic of cytokinin action, were accentuated also of those bud cultures which had been treated with BAP or with CFM. (2) In the callus cultures, the cytokinin characteristics appeared under CFM influence in the peroxidase patterns of one of the tissue culture strains only when CFM was applied in combination with BAP and not in combinations of CFM with IAA.The observed morphactin-induced increase in the cytokinin effects could occur via changes in the hormone level of the tissue.     

Amaranthin accumulation in continuous red and blue light by seedlings ofAmaranthus caudatus L.

Four days oldAmaranthus seedlings responded to light treatment with an increase of amaranthin accumulation. With increasing irradiation time, red light caused a saturation effect. Blue light induced a high irradiation response. The blue light effect was reversible to a certain extent by far-red irradiation given at the end of the treatment with blue light. Intermittent red light (3 h red light, 3 h dark, …) caused a higher amaranthin accumulation than 24 h continuous red light. Results obtained with red and blue light are discussed on the basis of the phytochrome system.     

Leucine interference in the production of water-soluble red Monascus pigments

The formation of soluble Monascus red pigments is strongly positively and negatively regulated by different amino acids. Leucine, valine, lysine, and methionine had strong negative effects on pigment formation. Leucine supported poor pigment formation when used as sole nitrogen source in fermentations, yet it neither repressed pigment synthase(s) nor inhibited its action. The new pigments derived from the hydrophobic leucine were more hydrophilic than the conventional red pigments (lacking an amino acid side-chain) and were extracellularly produced. Therefore, the low level of red pigments produced when leucine was the nitrogen source was not due to feed-back regulation by cell-bound leucine pigments. The negative effect of leucine was caused by enhanced decay of pigment synthase(s). The enhanced decay was not due simply to de novo synthesis of a leucine-induced protease.Abbreviations mSG Monosodium glutamate - MOPS 3-(N-morpholine)propane sulfonic acid - DCW dry cell weight