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.     

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     

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.     

Interaction of a radiolabeled cytokinin photoaffinity probe with a receptor protein

A photoreactive analog of the cytokinin 6-benzylaminopurine was prepared by the method of Theiler $\text{et. al.}$ (1) modified so as to include a radioactive atom in the final product, [methylene-¹⁴C] 2-azido-6-benzylaminopurine. The affinity of this doubly labeled cytokinin probe for a previously described cytokinin receptor protein (2,3) is very nearly the same as for the parent cytokinin. The cytokinin probe was covalently incorporated into the receptor protein by irradiation with ultraviolet light, and its presence there was quantitatively established by assaying for non-dialyzable ¹⁴C. The labeled protein was subjected to SDS polyacrylamide gel electrophoresis and the subunits assayed for radioactivity by fluorography. Each of the four subunits of the receptor protein was labeled with ¹⁴C to some extent. The data suggest that all four subunits of the protein either actively participate in the formation of the cytokinin binding site or exist in close proximity to it.     

Two effects of cytokinin on the auxin requirement of tobacco callus cultures

Cytokinin affects the requirement for auxin of a strain of tobacco callus (Nicotiana tabacum) which is cytokinin-autotrophic when grown on Murashige and Skoog medium with 11.4 mum of indole-3-acetic acid but requires cytokinin 6-(3-methyl-2-butenylamino)purine (i(6) Ade) when grown on the same medium with <3 mum indole-3-acetic acid. As the exogenous concentration of cytokinin (i(6) Ade) is increased, the concentration of indole-3-acetic acid required for growth is decreased. A second effect of cytokinin, observed sporadically in cultures with 2.5 mum or 5 mum i(6) Ade, is the transformation of some of the callus pieces to auxin-autotrophic growth. Strains, both callus-forming and bud-forming tissues, that arise in this manner are not permanently altered in their auxin requirement because subcultures on medium without cytokinin still require exogenous auxin.     

Physiology and biochemistry of theAmaranthus cytokinin bioassay and its applications

The influence of some substances on the amaranthin biosynthesis inAmaranthus-seedlings was investigated with regard to the accuracy and sensitivity of theAmaranthus cytokinin bioassay. 1) The herbicide glyphosate inhibited plgment formation in a concentration of 10^-4 mol 1^-1 in most cases only slightly (approximately 10–25 %). 2) Shikimic acid does not seem to be a precursor, in many experiments it was slightly inhibitory. 3) Jasmonic acid promoted slightly the plgment formation with a distinct maximum at about 5 x 10^-4 mol 1^1-. 4) The oxidation product of zeatin (with KMnO₄) is not active in theAmaranthus bioassay; only weak activity was recorded at very high concentrations. 5) Glucosides, extracted fromGinkgo biloba leaves, stimulate amaranthin synthesis, as we could conclude from applications of this bioassay to senescing, abscisingGinkgo leaves. Presented at the International Symposium “Plant Growth Regulators” held on June 18–22, 1984 at Liblice, Czechoslovakia.     

Influence of Different Cytokinins on the Transpiration and Senescence of Excised Oat Leaves

In order to investigate the possibility that cytokinins control transpiration indirectly through affecting leaf senescence, a direct comparison was made of the effect of different cytokinins on transpiration and senescence of oat leaves (Avena sativa L. cv. Forward). Senescence was assessed by measuring chlorophyll loss. The synthetic cytokinins N⁶ benzyladenine (BA) and kinetin delayed senescence and increased transpiration of oat leaves to a greater extent than did the naturally occurring compounds zeatin, N^b-Δ² isopentenyladenine (i⁶ Ade) and 6-ø-hydroxybenzyladenosine (hyd-BA riboside). During the early stages of the transpiration experiment zeatin showed similar or greater activity than BA. This period was longest when freshly excised leaves were used, was reduced when leaves were used after incubation in distilled water in the dark for 20 h and was eliminated by incubation in cytokinin solution in the dark. After this period the activity of zeatin declined relative to BA. The effect of cytokinins in increasing transpiration occurred only in the light; no effect was observed in the dark. BA showed higher activity than zeatin in senescence tests but both cytokinins were less effective as the tests progressed, this decrease in activity being more rapid when older leaves were used. The results are discussed in relation to the mechanisms by which endogenous cytokinins might control sensecence and transpiration in oat leaves and to the value of the oat leaf senscence and transpiration bioassays as tests for cytokinin activity of plant extracts.     

Light-induced expression of ipt from Agrobacterium tumefaciens results in cytokinin accumulation and osmotic stress symptoms in transgenic tobacco

Cytokinins are plant growth regulators that induce shoot formation, inhibit senescence and root growth. Experiments with hydroponically grown tobacco plants, however, indicated that exogenously applied cytokinin led to the accumulation of proline and osmotin. These responses were also associated with environmental stress reactions, such as salt stress, in many plant species. To test whether increased endogenous cytokinin accumulation led to NaCl stress symptoms, the gene ipt from Agrobacterium tumefaciens, encoding isopentenyl transferase, was transformed into Nicotiana tabacum cv. SR-1 under the control of the light-inducible rbcS-3A promoter from pea. In high light (300 mol PPFD m^-2 s^-1), ipt mRNA was detected and zeatin/zeatin glucoside levels were 10-fold higher than in control plants or when transformants were grown in low light (30 mol PPFD m^-2 s^-1). High light treatment was accompanied by increased levels of proline and osmotin when compared to low light grown transformed and untransformed control plants. Elevated in planta cytokinin levels induced responses also stimulated by salt stress, suggesting either common or overlapping signaling pathways are initiated independently by cytokinin and NaCl, setting in motion gene expression normally elicited by developmental processes such as flowering or environmental stress.Abbreviations IPT isopentenyl, transferase - rbcS-3A gene encoding a small subunit protein (SSU) of Rubisco from Pisum sativum - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

Natural variation in cytokinin maintenance improves salt tolerance in apple rootstocks

Plants experiencing salt‐induced stress often reduce cytokinin levels during the early phases of stress‐response. Interestingly, we found that the cytokinin content in the apple rootstock “robusta” was maintained at a high level under salt stress. Through screening genes involved in cytokinin biosynthesis and catabolism, we found that the high expression levels of IPT5b in robusta roots were involved in maintaining the high cytokinin content. We identified a 42 bp deletion in the promoter region of IPT5b, which elevated IPT5b expression levels, and this deletion was linked to salt tolerance in robusta×M.9 segregating population. The 42 bp deletion resulted in the deletion of a Proline Response Element (ProRE), and our results suggest that ProRE negatively regulates IPT5b expression in response to proline. Under salt stress, the robusta cultivar maintains high cytokinin levels as IPT5b expression cannot be inhibited by proline due to the deletion of ProRE, leading to improve salt tolerance.     

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene

The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with ³H and auxin was found to promote conversion of zeatin-type cytokinins to ³H-labelled adenine derivatives. When the very rapid metabolism of exogenous [³H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.Abbreviations Ade adenine - Ados adenosine - BA 6-benzylaminopurine - C control - Con A concanavallin A - CP cellulose phosphate - IPT isopentenyl transferase - NAA 1-naphthylacetic acid - NP normal phase - NPPU N-(3-nitrophenyl)-N-phenylurea - RIA radioimmunoassay - RP reversed phase We wish to thank Dr. J. Zwar for supplying phenylurea derivitives.     

Regulation of cytokinin oxidase activity in tobacco callus expressing the T-DNA ipt gene

There are indications that the cytokinin content in transgenic tissues expressing the cytokinin biosynthetic ipt gene is under metabolic control, which prevents the accumulation of cytokinins to lethal levels. The objective of this study was to investigate the relationships between the content of endogenous cytokinins and the activity of cytokinin oxidase (which is believed to be a copper-containing amine oxidase, EC 1.4.3.6.) in ipt transgenic tobacco callus. In addition, the effect of exogenously applied N-benzyladenine (BA) on this relationship was examined. Endogenous cytokinin concentrations were measured in callus of Nicotiana tabacum L. cv. Petit Havana SRI transformed with the ipt of Agrobacterium tumefaciens under the control of a light-inducible promoter and in non-transformed tissue using LC-tandem mass spectrometry. The activity of cytokinin oxidase was estimated by measuring the conversion of [2,8-³H]N⁶-(Δ²-isopentenyl)adenine to [³H]adenine by enzyme preparations in vitro. The 14-day-old ipt-transformed callus contained a 25-fold higher amount of cytokinins as compared to the non-transformed tissue. Mainly zeatin- and dihydrozeatin-types of cytokinins (free bases, ribosides, nucleotides and O-glucosides) accumulated in the ipt transgenic tissue. The cytokinin pool of both ipt-transformed and non-transformed tissues consisted predominantly of cytokinins that are either resistant to cytokinin oxidase attack (nucleotides and O-glucosides of cytokinins and cytokinins bearing N⁶-saturated side chain) or have a low affinity for the enzyme (zeatin and its riboside). The former represented 71.6 and 74.8% and the latter 27.7 and 24.4% of the pool of endogenous cytokinins in ipt-transformed and non-transformed tissues, respectively. Enzyme preparations from ipt-transformed tissue exhibited 1.5-fold higher cytokinin oxidase activity compared with that observed in control tissues. Application of exogenous BA affected the total levels of cytokinins of the two tissue lines in different ways. The cytokinin content increased by 1.7- and 1.5-fold in ipt-transformed tissues 6 and 12 h after BA application, respectively, while it declined in the non-transformed control by 1.6- to 2.0-fold between 3 and 12 h after BA application. The increase in cytokinin content in the ipt callus is due to an increase of zeatin- and dihydrozeatin-type cytokinins (nucleotides, ribosides and free bases) leading to an enhanced accumulation of O-glucosides after 12 h. Following BA treatment, the cytokinin oxidase activity increased up to 1.8-fold in ipt-transformed and 1.6-fold in non-transformed tissues. The levels of isopentenyl-type cytokinins were near the detection limit; however, the enhancement of cytokinin oxidase activity after BA treatment in both tissue lines was correlated with the content of preferred substrate of the enzyme, N⁶-(Δ²-isopentenyl)adenosine.     

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     

Kinetic comparison of cytokinin nucleosidase activity isolated from normally ripening and mutant tomato varieties

Kinetic parameters for cytokinin nucleosidase activity which catalyzes the deribosylation of N⁶(Δ²-isopentenyl)adenosine (I⁶Ado) to produce the more “active” free base N⁵(Δ²-isopenetyl)adenine (I⁶Ade) were compared for a normally ripening tomato (Lycopersicon esculentum L.) cultivar Rutgers, and two mutant tomato varieties (Nor and Rin). K_m for nucleosidase activity in Rutgers was lower (K_m = 0.1 millimolar) than that in either Nor (K_m = 0.14 millimolar) or Rin (K_m = 0.13 millimolar).     

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.     

Cytokinin Oxidase from Phaseolus vulgaris Callus Tissues : Enhanced in Vitro Activity of the Enzyme in the Presence of Copper-Imidazole Complexes

The effects of metal ions on cytokinin oxidase activity extracted from callus tissues of Phaseolus vulgaris L. cv Great Northern have been examined using an assay based on the oxidation of N⁶-(Δ²-isopentenyl)-adenine-2,8-³H (i⁶ Ade) to adenine (Ade). The addition of cupric ions to reaction mixtures containing imidazole buffer markedly enhanced cytokinin oxidase activity. In the presence of optimal concentrations of copper and imidazole, cytokinin oxidase activity was stimulated more than 20-fold. The effect was enzyme dependent, specific for copper, and observed only in the presence of imidazole. The substrate specificity of the copper-imidazole enhanced reaction, as judged by substrate competition tests, was the same as that observed in the absence of copper and imidazole. Similarly, in tests involving DEAE-cellulose chromatography, elution profiles of cytokinin oxidase activity determined using a copper-imidazole enhanced assay were identical to those obtained using an assay without copper and imidazole. On the basis of these results, the addition of copper and imidazole to reaction mixtures used to assay for cytokinin oxidase activity is judged to provide a reliable and specific assay of greatly enhanced sensitivity for the enzyme. The mechanism by which copper and imidazole enhance cytokinin oxidase activity is not certain, but the reaction catalyzed by the enzyme was not inhibited by anaerobic conditions when these reagents were present. This observation suggests that copper-imidazole complexes are substituting for oxygen in the reaction mechanism by which cytokinin oxidase effects cleavage of the N⁶-side chain of i⁶Ade.     

The synthesis and biological properties of 8-azido-N 6-benzyladenine,a potential photoaffinity reagent for cytokinin

A cytokinin photoaffinity reagent, 8-azido-N ⁶-benzyladenine (8N₃BA), was synthesized from 8-bromoadenosine via azide replacement, benzylation at N–1, rearrangement to the N-6-benzyl derivative and acid hydrolysis. The compound thus obtained was found to have full cytokinin activity in the moss and tobacco cell-suspension bioassays. Photolysis of 8N₃BA was accomplished with long and short-wavelength ultraviolet light and produced compounds which had very little or no biological activity in the two bioassays. In-vivo photolysis of 8N₃BA caused loss of the cytokinin activity of this compound in moss protonemata. This result was similar to earlier ones where the biological response of moss protonemata to benzyladenine was reversed following removal of the hormone by a short rinse with water.Abbreviations BA N ⁶-benzyladenine - 8N₃BA 8-azido-N ⁶-benzyladenine - PMR proton magnetic resonance - TLC thin-layer chromatography - UV ultraviolet In partial fulfillment of requirements for the Ph.D. degree at Michigan State University     

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.