Substituted nitroguanidines provide cytokinin activity during in vitro cultivation of plant tissues

Synthetic nitroguanidine derivatives can be used as alternatives to the traditional adenine-containing cytokinins used in plant tissue culture. First, nitroguanidine derivatives (NG) mimicked the typical activity of two standard cytokinins, 6-benzylaminopurine (BAP) and 2-isopentenyladenine (2iP) in the soybean callus (Glycine max) growth bioassay. NGs caused unanticipated responses as well, as demonstrated in three lines of tobacco (Nicotiana tabacum), when the auxin concentration was reduced from the standard concentration of 2 ug/ml NAA, to much lower concentrations of 0.01 ug/ml NAA or 0.02 ug/ml IAA. At the low auxin concentrations, kinetin lost the ability to promote either growth or differentiation, while the NG cytokinins were fully able to promote both. NGs promoted growth and differentiation in the presence of 0.01 ug/ml NAA in a newly initiated, totipotent line of Coker 319 tobacco. NGs plus 0.02 ug/ml IAA also promoted callus growth in a cytokinin-habituated tobacco line, Havana 425-CH. Lastly, NGs stimulated the outgrowth of healthy callus from aged callus that had been allowed to deteriorate through lack of subculture. Upon transfer of aged NTP callus to fresh media with NGs and 0.02 ug/ml IAA, healthy cell clusters were rapidly produced. In all three cases cited above, kinetin was ineffective at the low auxin concentrations. The NGs are therefore cytokinins, with the additional possibility of reducing the level of auxin required for their activity to be expressed.Abbreviations BAP 6-benzylaminopurine or N⁶- benzyladenine - IAA indoleacetic acid - 2iP N⁶-(²- isopentenyl)adenine - NAA -naphthaleneacetic acid - NG nitroguanidine derivative     

Analysis of gametophytic development in the moss,Physcomitrella patens,using auxin and cytokinin resistant mutants

Mutants altered in their response to auxins and cytokinins have been isolated in the moss Physcomitrella patens either by screening clones from mutagenized spores for growth on high concentrations of cytokinin or auxin, in which case mutants showing altered sensitivities can be recognized 3–4 weeks later, or by non-selective isolation of morphologically abnormal mutants, some of which are found to have altered sensitivities. Most of the mutants obtained selectively are also morphologically abnormal. The mutants are heterogeneous in their responses to auxin and cytokinin, and the behaviour of some is consistent with their being unable to make auxin, while that of others may be due to their being unable to synthesize cytokinin. Physiological analysis of the mutants has shown that both endogenous auxin and cytokinin are likely to play important and interdependent roles in several steps of gametophytic development. Although their morphological abnormalities lead to sterility, genetic analysis of some of the mutants has been possible by polyethyleneglycol induced protoplast fusion.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - NAA 1-naphthalene acetic acid - 2,4D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - IAP 6-( ²isopentenyl) aminopurine - NAR NAA resistant mutants - BAR BAP resistant mutants     

Cytokinin-induced hypocotyl elongation in light-grown<Emphasis Type="Italic"> Arabidopsis</Emphasis> plants with inhibited ethylene action or indole-3-acetic acid transport

Cytokinins inhibit hypocotyl elongation in darkness but have no obvious effect on hypocotyl length in the light. However, we found that cytokinins do promote hypocotyl elongation in the light when ethylene action is blocked. A 50% increase in Arabidopsis thaliana (L.) Heynh. hypocotyl length was observed in response to N⁶-benzyladenine (BA) treatment in the presence of Ag⁺. The level of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid was strongly increased, indicating that ethylene biosynthesis was up-regulated by treatment with cytokinin. Furthermore, the effects of cytokinins on hypocotyl elongation were also tested using a series of mutants in the cascade of the ethylene-signal pathway. In the ethylene-insensitive mutants etr1-3 and ein2-1, cytokinin treatment resulted in hypocotyl lengths comparable to those of wild-type seedlings treated with both Ag⁺ and BA. A similar phenotypical response to cytokinin was observed when auxin transport was blocked by -naphthylphthalamic acid (NPA). Applied cytokinin largely restored cell elongation in the basal and middle parts of the hypocotyls of NPA-treated seedlings and at the same time abolished the NPA-induced decrease in indole-3-acetic acid levels. Our data support the hypothesis that, in the light, cytokinins interact with the ethylene-signalling pathway and conditionally up-regulate ethylene and auxin synthesis.     

The control of bud dormancy in potato tubers. Measurement of the seasonal pattern of changing concentrations of zeatin-cytokinins

A radioimmunoassay, combined with high-performance liquid chromatography, has been used to analyse the zeatin-type cytokinins of potato (Solanum tuberosum L. cv. Majestic) tubers and tuber buds throughout growth and storage. During tuber growth, zeatin riboside was the predominant cytokinin detected in all tissues. Immediately after harvest, the total cytokinin concentration fell dramatically in the storage tissue, largely as a consequence of the disappearance of zeatin riboside. During storage, levels of cytokinins in the storage tissue remained relatively constant, but increased in the tuber buds. In the buds of tubers stored at 2°C there was a 20-to 50-fold increase in total cytokinin over six weeks, coinciding with the natural break of innate dormancy. At 10°C the rise in the level of bud cytokinins was slower, correlating with the longer duration of innate dormancy. Injecting unlabelled cytokinins into tubers in amounts known to induce sprouting gave rise to increases in cytokinin concentrations in the buds of the same order as the increase associated with the natural break of dormancy. Metabolism of injected cytokinins was greater in non-dormant than in dormant tubers. The roles of cytokinin concentration and the sensitivity of the buds to cytokinin in the control of dormancy are discussed.Abbreviations CK cytokinin - FW fresh weight - HPLC high-performance liquid chromatography - RIA radioimmunoassay - tio⁶ade 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-purine=zeatin - tio⁶adeglc⁹ 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-glucopyranosyl purine=zeatin-9-glucoside - tio⁶ado 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-ribofuranosyl purine=zeatin riboside - tio⁶ado-[³H]-diol a radioactive derivative of zeatin riboside, synthesised by periodate-oxidation followed by [³H]NaBH₄-reduction - tio⁶AMP 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-5-phosphoribofuranosyl purine=zeatin riboside 5-monophosphate - t(ioglc⁴)⁶ade 6-(4-O--D-glucopyranosyl-3-methylbut-trans-2-enylamino)-purine=zeatin-O-glucoside     

Mass-spectrometric quantification of cytokinin nucleotides and glycosides in tobacco crown-gall tissue

the cytokinins of tobacco crown-gall tissue have been analysed by quantitative mass spectrometry using ²H₂-labelled cytokinin riboside 5-monophosphates and ¹⁵N₄-labelled cytokinin glycosides as internal standards. The principal endogenous cytokinin of this tissue is zeatin riboside 5-monophosphate. The biologically inactive 7-glucoside of zeatin is the most abundant basic cytokinin in the tissue. These findings expose the limitations of previously reported analyses of similar tissues, which were restricted to biologically active basic cytokinins. The present study demonstrates that the endogenous cytokinins of tobacco crowngall tissue show a clear correspondence to the range of metabolites formed when exogenous cytokinins are supplied to nontumorous tobacco cells.Abbreviations DHZ dihydrozeatin - DHZ7G dihydrozeatin 7-glucoside - DHZMP dihydrozeatin 9-riboside 5-monophosphate - DHZR dihydrozeatin 9-riboside - GC-MS coupled gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - Z7G zeatin 7-glucoside - Z9G zeatin 9-glucoside - ZOG zeatin O-glucoside - ZMP zeatin 9-riboside 5-monophosphate - ZR zeatin 9-riboside - ZROG zeatin 9-riboside O-glucoside     

Regenerative Capacity of Cacti Schlumbergera and Rhipsalidopsis in Relation to Endogenous Phytohormones, Cytokinin Oxidase/Dehydrogenase, and Peroxidase Activities

The recalcitrant nature and increased regenerative capacity in relation to in vitro subcultures in two cactus genera Rhipsalidopsis (Easter cactus) and Schlumbergera (Christmas cactus) were studied by examining the endogenous concentrations of several endogenous phytohormones and enzyme activities. Leaf tissue from greenhouse-grown mother plants, in vitro subcultures 1 and 3, and callus tissues were analyzed and correlated with regenerative ability. The cytokinins present in the two cacti genera were mainly isopentenyl-type derivatives. The total content of isopentenyl-type cytokinins in greenhouse-grown leaves of Rhipsalidopsis was more than twice the amount found in greenhouse-grown leaves of Schlumbergera. The total cytokinin content decreased during subculturing. Cytokinin oxidase/dehydrogenase (CKX, EC 1.4.3.18/1.5.99.12) activity increased during subculturing. In Schlumbergera there is no effect of subculturing on CKX and related cytokinin homeostasis. The total peroxidase (POX, EC 1.11.1.7) activity in greenhouse-grown leaves of both genera was low, and the activity increased significantly during subculturing, more specifically in the tissue of Rhipsalidopsis. The results clearly indicated that an enhanced auxin metabolism (biosynthesis, conjugation/deconjugation, and POX activity), in combination with an enhanced CKX activity, shifts the auxin and cytokinin pool, favoring adventitious shoot formation in Rhipsalidopsis, whereas the low level of POX activity, together with auxin autotrophy/conjugation, makes Schlumbergera more recalcitrant. S. S. and E. P. contributed equally to this work     

Cytokinin inhibition of respiration by cells and mitochondria of soybean,Glycine max (L.) Merrill

Cells of a soybean tissue strain, suspended in an aerated liquid medium, caused disappearance of p-coumaric acid from the medium and oxidation of guaiacol, benzidine, pyrogallol, L-dihydroxyphenylalanine and L-epinephrine. Both the disappearance and the oxidations were inhibited by 6-benzylaminopurine (BAP) at a concentration of 0.5 mM. BAP at other concentrations either promoted or inhibited oxidation of epinephrine in precisely the pattern reported earlier for the disappearance of coumarate; therefore, the disappearance of coumarate probably involves its oxidation. The effectiveness of other cytokinins in inhibiting the oxidation was studied.At 0.5 mM, and perhaps even at 0.5 M, some of the several cytokinins tested inhibited oxygen consumption by the soybean cells. This inhibition, which did not require any of the above metabolizable compounds, was especially marked in the presence of cyanide, azide or Antimycin A, and was detectable in 10 min or less. Either Antimycin A or salicylhydroxamic acid alone promoted O₂ consumption but together they were quite inhibitory. The soybean cells apparently have an alternate respiratory pathway and cytokinins may influence its operation.Several cytokinins at 0.5 mM, and perhaps at 0.5 M, also inhibited oxygen consumption by mitochondrial preparations from the soybean cells, the inhibition being evident in about 20 s. The consumption required a substrate such as malate, succinate or NADH. Cytokinins and related compounds varied in effectiveness as follows: BAP and 6-isopentenyla-minopurine 9-tetrahydropyranyl-BAP > kinetin, ribosyl-isopentenylaminopurine, 9-methyl-BAP and 9-methoxymethyl-BAP > 6,6-dimethylaminopurine and zeatin (slight activity) > 6-methylaminopurine, nicotinamide and adenine (ineffective). To a great extent this order parallels the order of effectiveness of the compounds in causing cell division. Mitochondria, therefore, may contain a site for an important cytokinin action.Abbreviations BAP 6-benzylaminopurine - IPA 6-(²-isopentenyl)aminopurine     

The biosynthesis of cytokinins in crown-gall tissue of Vinca rosea

The crown-gall tissue of Vinca rosea converts labelled adenine into cytokinins. The principal initial products appear to be ribosylzeatin phosphates; zeatin and ribosylzeatin are also produced in appreciable quantities. The efficiency of conversion of adenine into cytokinins suggests a pathway of synthesis independent of turnover of tRNA. Isopentenyl adenine or its derivatives do not appear to be intermediates in the conversion of adenine to zeatin compounds. Cytokinins in V. rosea turnover rapidly and further metabolism of zeatin derivatives seems to result in their conversion into glucosides which are the main cytokinin active compounds in the tissue.Abbreviations HPLC high performance liquid chromatography - AMP adenosine monophosphate - TLC thin-layer chromatography - GLC gas-liquid chromatography     

The effect of auxin concentration on cytokinin stability and metabolism

The stability of [³H]zeatin riboside supplied to freshly excised tobacco pith explants was found to be inversely related to -naphthaleneacetic acid concentration in the incubation medium. At higher concentrations of -naphthaleneacetic acid greater breakdown of [³H]zeatin riboside was indicated by higher levels of degradative metabolites (adenine, adenosine and adenosine nucleotides) formed. This auxin effect on cytokinin metabolism appears to be mediated, at least in part, through cytokinin oxidase. The results of in-vitro assays carried out with partially purified enzyme from corn kernels substantiale this conclusion. These findings are discussed in relation to recent observations of auxin and cytokinin levels in crown-gall tumours with altered morphology.Abbreviations FPLC fast protein liquid chromatography - HPLC high-performance liquid chromatography - IP isopentenyladenine - NAA naphthaleneacetic acid - ZR zeatin riboside     

Some effects of plant growth regulators on tissue cultures of the marine red alga Agardhiella subulata (Gigartinales,Rhodophyta)

We examined whether auxins and cytokinins, either singly or in combination, stimulate cell division in tissue cultures of a red seaweed. Our experimental model consisted of filamentous and callus-like growths that developed from cross-sectional discs cut from young branches of Agardhiella subulata. Plant growth regulators were added to the medium to give combinations of an auxin with a cytokinin over a range of concentrations (1 µg L^–1 –10 mg L^–1). Several mixtures of auxins and cytokinins, as well as some single auxins, cytokinins and phenolics, stimulated cell division and growth in the tissue cultures beyond that of controls. The treatments that were effective included: phenylacetic acid/zeatin; phenylacetic acid/6-benzylaminopurine; -naphthaleneacetic acid/zeatin; 2,4,5-trichlorophenoxyacetic acid/6-benzylaminopurine; and indoleacetic acid/kinetin. High concentrations of cytokinins (i.e. 10 mg L^–1) inhibited the regeneration of plants in some of the cell cultures. These results provide further evidence that growth regulators can be used for the tissue culture of seaweeds and for the study of developmental phenomena in these plants.     

Expression of the isopentenyl transferase gene is regulated by auxin in transgenic tobacco tissues

The isopentenyl transferase gene (ipt) fromAgrobacterium tumefaciens was isolated and introduced, via a disarmed binary vector, into tobacco using theAgrobacterium tumefaciens-mediated gene transfer system. The expression of theipt gene was monitored by RNA hybridization, western blotting and cytokinin analysis. The addition of auxin to the media rapidly reduced the level of cytokinins in the transgenic tissues and this was associated with a reduction in IPT mRNA and protein levels. It is concluded that the hormone auxin can regulate expression of a gene involved in biosynthesis of the second hormone cytokinin. Although exogenous benzyladenine did not directly affectipt gene expression, it did antagonize the effect of auxin on levels of cytokinins and IPT mRNA and protein.     

The role of cellulase in hormonal regulation of shoot morphogenesis in tobacco callus

A polyclonal antibody raised against cellulase (EC 3.2.1.4.) from callus ofNicotiana tabacum L. cv. Petit Havana SR1 reduced cellulase activity and induced shoot formation in tobacco callus in the presence of callus maintaining concentrations of auxin and cytokinin. Shoot induction as well as reduction of the cellulase activity was also obtained by withdrawing auxin from the callus medium. The effect of the two hormones on cellulase activity in the tobacco tissue was examined by varying the concentration of one of the hormones -naphthylacetic acid (NAA) or benzylaminopurine (BAP) at a time while the other was kept at a level sufficient for either callus growth or shoot induction. While NAA stimulated the enzyme activity increasingly with concentration in the range 5 × 10^–7 M to 5 × 10^–5 M at both levels of BAP, BAP only stimulated the cellulase activity at an optimum concentration of 5 × 10^–6 M when NAA was present at a level sufficient to induce callus growth. The results point to a pivotal role of the downward regulation of cellulase in the initiation of shoot induction. A series of events leading to oriented cell divisions as a result of the lowered cellulase level during the initial phase of the morphogenetic process is discussed.Abbreviations Ab Purified cellulase antibody - BAP benzylaminopurine - MS Murashige and Skoog medium - NAA -naphthylacetic acid - PS Purified preimmune serum We thank Mr. Poul Fabech for constructing the automatic viscosimetric equipment and Mr. Hans Hjorth for making the computer programme. This work was supported by The Danish Veterinary and Agricultural Research Council.     

The Involvement of Cytokinin Oxidase/Dehydrogenase and Zeatin Reductase in Regulation of Cytokinin Levels in Pea (Pisum sativum L.) Leaves

Cytokinin metabolism in plants is very complex. More than 20 cytokinins bearing isoprenoid and aromatic side chains were identified by high performance liquid chromatography-mass spectrometry (HPLC-MS) in pea (Pisum sativum L. cv. Gotik) leaves, indicating diverse metabolic conversions of primary products of cytokinin biosynthesis. To determine the potential involvement of two enzymes metabolizing cytokinins, cytokinin oxidase/dehydrogenase (CKX, EC 1.5.99.12) and zeatin reductase (ZRED, EC 1.3.1.69), in the control of endogenous cytokinin levels, their in vitro activities were investigated in relation to the uptake and metabolism of [2−³H]trans-zeatin ([2−³H]Z) in shoot explants of pea. Trans-zeatin 9-riboside, trans-zeatin 9-riboside-5′-monophosphate and cytokinin degradation products adenine and adenosine were detected as predominant [2−³H]Z metabolites during 2, 5, 8, and 24 h incubation. Increasing formation of adenine and adenosine indicated extensive degradation of [2−³H]Z by CKX. High CKX activity was confirmed in protein preparations from pea leaves, stems, and roots by in vitro assays. Inhibition of CKX by dithiothreitol (15 mM) in the enzyme assays revealed relatively high activity of ZRED catalyzing conversion of Z to dihydrozeatin (DHZ) and evidently competing for the same substrate cytokinin (Z) in protein preparations from pea leaves, but not from pea roots and stems. The conversion of Z to DHZ by pea leaf enzyme was NADPH dependent and was significantly inhibited or completely suppressed in vitro by diethyldithiocarbamic acid (DIECA; 10 mM). Relations of CKX and ZRED in the control of cytokinin levels in pea leaves with respect to their potential role in establishment and maintenance of cytokinin homeostasis in plants are discussed.     

Purification and identification of a cytokinin from moss callus cells

A cytokinin was isolated from the culture medium of callus cells of the moss hybridFunaria hygrometrica (L.) Sibth xPhyscomitrium piriforme Brid. The purification procedure included ethyl-acetate extraction, silver-salt precipitation, crystallization as picrate, and ion exchange chromatography. The structure of the cytokinin was confirmed as N⁶–(²-isopentenyl)adenine by means of gas chromatography and mass spectrometry. The concentration of the compound in the culture medium was determined at ca. 10^-6 M.Abbreviation 2iP N⁶–(²-isopentenyl) adenine     

Biochemical Characterization of Cytokinin Oxidases/Dehydrogenases from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Expressed in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L.

Transgenic tobacco plants overexpressing single Arabidopsis thaliana cytokinin dehydrogenase (CKX, EC 1.5.99.12) genes AtCKX1, AtCKX2, AtCKX3, AtCKX4, AtCKX5, AtCKX6, and AtCKX7 under the control of a constitutive 35S promoter were tested for CKX-enzymatic activity with varying pH, electron acceptors, and substrates. This comparative analysis showed that out of these, only AtCKX2 and AtCKX4 were highly active enzymes in reaction with isoprenoid cytokinins (N ⁶ -(2-isopentenyl)adenine (iP), zeatin (Z)) and their ribosides using the artificial electron acceptors 2,6-dichlorophenol indophenol (DCPIP) or 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q₀). Turnover rates of these cytokinins by four other AtCKX isoforms (AtCKX1, AtCKX3, AtCKX5, and AtCKX7) were substantially lower, whereas activity of AtCKX6 was almost undetectable. The isoenzymes AtCKX1 and AtCKX7 showed significant preference for cytokinin glycosides, especially N ⁶ -(2-isopentenyl)adenine 9-glucoside, under weakly acidic conditions. All enzymes preferentially cleave isoprenoid cytokinins in the presence of an electron acceptor, but aromatic cytokinins are not resistant and are degraded with lower reaction rates as well. Cytokinin nucleotides, considered as resistant to CKX attack until now, were found to be potent substrates for some of the CKX isoforms. Substrate specificity of AtCKXs is discussed in this study with respect to the structure of the CKX active site. Further biochemical characterization of the AtCKX1, AtCKX2, AtCKX4 and AtCKX7 enzymes showed pH-dependent activity profiles.     

Response of cytokinin concentration in the xylem exudate of bean (Phaseolus vulgaris L.) plants to decapitation and auxin treatment,and relationship to apical dominance

When xylem exudate of previously untreated Phaseolus vulgaris plants was analysed for cytokinins by radioimmunoassay, a low concentration (about 5 ng · ml^–1) was found. However, when the plants were decapitated about 16 h before the xylem exudate was collected, an almost 25-fold increase in cytokinin concentration was observed. Twenty-four hours after decapitation this increase even reached 4000 compared to control plants. Applying naphthaleneacetic acid (NAA) to the shoot of decapitated plants almost eliminated the effect of shoot tip removal on cytokinin concentration, suggesting that cytokinins in the xylem exudate of intact plants are under the control of the polar auxin transport system. Other xylem constituents, such as potassium or free amino acids did not show this strong increase after decapitation and did not respond to NAA application. It is concluded that the observed auxin/cytokinin interaction has an important regulatory role to play, not only in apical dominance but in many other correlative events as well.Abbreviations AD apical dominance - CKs cytokinin(s) - iAde/iAdo isopentenyladenine/iospentenyladenosine - NAA naphthaleneacetic acid - Z/ZR zeatin/zeatin riboside     

Biosynthesis of cytokinins

Cytokinins are adenine derivatives with an isoprenoid side chain and play an essential role in plant development. Plant isopentenyltransferases that catalyze the first and rate-limiting steps of cytokinin biosynthesis have recently been identified. Unlike bacterial enzymes, which catalyze the transfer of the isopentenyl moiety from dimethylallyldiphosphate (DMAPP) to the N ⁶ position of adenosine 5′-monophosphate (AMP), plant enzymes catalyze the transfer of the isopentenyl moiety from DMAPP preferentially to ATP and to ADP. The isopentenylated side chain is hydroxylated to form zeatin-type cytokinins. An alternative pathway, in which a hydroxylated side chain is directly added to the N ⁶ position of the adenine moiety, has also been suggested.     

Kinetin-induced stimulation of electrogenic pumping in soybean suspension cultures is unrelated to signal transduction

Primary modes of action of cytokinins have been thought to involve stimulation of the electrogenic H⁺ pump and-or opening of plasmamembrane Ca²⁺ channels. In order to test these hypotheses, rapid changes in membrane transport in response to cytokinin application were studied in heterotrophic suspension-cultured callus of soybean (Glycine max (L.) Merr.) using electrophysiological techniques. Kinetin (N⁶-furfurylaminopurine; 2 M) elicited membrane hyperpolarization of 13±1 mV. This effect occurred even at membrane poteintials more negative than the most negative ionic equilibrium potential, and therefore might have resulted either from stimulation of the electrogenic pump, or from closure of ionic channels. The former mechanism of action appears most likely because (i) kinetin-induced membrane hyperpolarization is not accompanied by a significant change in plasma-membrane resistivity and (ii) hyperpolarization is abolished by cyanide, which inhibits electrogenic pump activity by depletion of cellular ATP.Electrogenic pumping is also activated by two other cytokinins: N⁶-(benzyl)adenine and trans-zeatin. However, it is unlikely that the hormonal effect on electrogenesis is directly related to transduction of the cytokinin signal, for the following reasons: (i) hormonally inactive, but chemically related compounds (cis-zeatin, adenine) also elicited membrane hyperpolarization; (ii) hormonally active, N⁹-substituted cytokinins failed to stimulate electrogenesis; (iii) the chemically unrelated cytokinin N,N-diphenylurea also failed to stimulate electrogenesis.The results imply that the kinetin effect on electrogenic pumping is related to adenine, or its metabolism, and not hormonal action. Adenine was absorbed by soybean cells, but not in sufficient quantities to have a significant effect on adeninenucleotide pools. It appears likely that the control of electrogenesis requires either the presence of a purine free base (i.e. no substituents at the N⁹ position) or phosphoribosylation of the free base. No evidence was found for cytokinin-induced Ca²⁺-channel opening, though it is argued that such an event might be physiologically relevant, yet undetectable with the methods employed. It is essential that future studies on cytokinin signal transduction — especially as they relate to membrane transport — take into account the possibility that metabolic effects unrelated to hormone action are dominant.Abbreviations and symbols bzl⁶Ade N⁶ (benzyl)adenine - SRB Soybean Recording Buffer - V_m membrane potential     

Effect of exogenous cytokinins,auxins and adenine on cytokinin <Emphasis Type="Italic">N</Emphasis>-glucosylation and cytokinin oxidase/dehydrogenase activity in de-rooted radish seedlings

The role of cytokinin N-glucosylation and degradation by cytokinin oxidase/dehydrogenase (CKX, EC 1.5.99.12) in response to application of exogenous auxins (2,4-dichlorophenoxyacetic acid [2,4-D] and -naphthaleneacetic acid [NAA]) and cytokinins (N ⁶-benzyladenine [BA] and trans-zeatin [Z]) was investigated in de-rooted seedlings of Raphanus sativus L. cv. Rampouch. Both auxins applied for 24 h at 1 and 10 M concentration increased N-glucosylation of exogenously applied [³H]dihydrozeatin (DHZ) by up to 20%. The level of endogenous 7N-glucosides (of Z, isopentenyladenine [iP] and DHZ) was increased by 2,4-D and NAA at 10 M concentration by 28 and 23%, respectively, the level of Z being decreased by 90 and 59%, respectively. 2,4-D and NAA suppressed CKX activity ca. by half. Exogenous cytokinins Z and BA applied at 1 and 10 M concentration stimulated 7N-glucosylation of [³H]DHZ (by up to 40%). BA both at 1 and 10 M, increased the level of endogenous Z by up to 35% and that of 7N-glucosides by up to 27%. BA application also strongly stimulated CKX activity (by up to 180%). Feeding with 1 and 10 M Z resulted in ca. 100-fold and 2000-fold increase of Z level, respectively. The main metabolite, Z7G, was increased ca. 6-fold and 60-fold, respectively. Levels of Z 9-glucoside (Z9G), trans-zeatin riboside (ZR) and Z O-glucoside (ZOG) were elevated to lesser extent. As compared to BA, Z had only negligible effect on CKX activity. Adenine (1–500 M) was preferentially 7N-glucosylated inhibiting competitively 7N-glucosylation of [³H]DHZ. At high concentrations (100–500 M) it increased endogenous levels of active cytokinins, especially of Z, however, it had no effect on CKX activity. Cytokinin N-glucosylation proved to be involved in down-regulation of active cytokinins in response to auxin and in the re-establishment of cytokinin homeostasis following application of exogenous cytokinins.