Regulators of cell division in plant tissues. XXX. Cytokinin metabolism in relation to radish cotyledon expansion and senescence

Kinetic studies of formation of glucosides of 6-benzylaminopurine (BAP) in excised radish cotyledons indicated that the 3-, 7-, and 9-glucosides (N-glucosides) were each formed directly from BAP. The 7- and 9-glucosides of BAP and the 7-glucoside of zeatin exhibited great stability in the cotyledons, but the 3-glucoside was converted to free BAP and to the 7- and 9-glucosides of BAP. When³H-labeled zeatin was supplied to developed cotyledons, at high concentrations (100 M), 7-glucosylzeatin was the principal metabolite, but an appreciable proportion of the extracted³H was due to O-glucosylzeatin. In immature cotyledons, as used in the radish cotyledon cytokinin bioassay, this O-glucoside was shown to be converted into zeatin 7-glucoside probably via free zeatin.Metabolism of BAP and zeatin in radish cotyledons was studied in relation to cytokinin-induced cotyledon expansion. Cytokinin N-glucosides were not metabolites responsible for the observed cytokinin-induced expansion, and were not detoxification products, or deactivation products formation of which was coupled with cytokinin action. However, the free base, its riboside, and nucleotide were possible active forms of BAP associated with cotyledon expansion. The possible significance of cytokinin N-glucosides is discussed.Senescent and nonsenescent cotyledons differed in their metabolism of BAP, zeatin, and zeatin riboside. Senescence was associated principally with a reduction in ability to form 7-glucosylzeatin, enhanced metabolism to adenine derivatives, and an inability to form appreciable amounts of 3-glucosyl-BAP.A two-dimensional thin layer chromatography (TLC) system, based on adjoining layers of cellulose and silica gel, for separating zeatin metabolites is described. This does not completely separate zeatin and zeatin riboside from the corresponding dihydro-compounds. A reversed phase TLC method for achieving these separations is also reported.     

Regulators of cell division in plant tissues. XXX. Cytokinin metabolism in relation to radish cotyledon expansion and senescence

Kinetic studies of formation of glucosides of 6-benzylaminopurine (BAP) in excised radish cotyledons indicated that the 3-, 7-, and 9-glucosides (N-glucosides) were each formed directly from BAP. The 7- and 9-glucosides of BAP and the 7-glucoside of zeatin exhibited great stability in the cotyledons, but the 3-glucoside was converted to free BAP and to the 7- and 9-glucosides of BAP. When³H-labeled zeatin was supplied to developed cotyledons, at high concentrations (100 μM), 7-glucosylzeatin was the principal metabolite, but an appreciable proportion of the extracted³H was due to O-glucosylzeatin. In immature cotyledons, as used in the radish cotyledon cytokinin bioassay, this O-glucoside was shown to be converted into zeatin 7-glucoside probably via free zeatin. Metabolism of BAP and zeatin in radish cotyledons was studied in relation to cytokinin-induced cotyledon expansion. Cytokinin N-glucosides were not metabolites responsible for the observed cytokinin-induced expansion, and were not detoxification products, or deactivation products formation of which was coupled with cytokinin action. However, the free base, its riboside, and nucleotide were possible active forms of BAP associated with cotyledon expansion. The possible significance of cytokinin N-glucosides is discussed. Senescent and nonsenescent cotyledons differed in their metabolism of BAP, zeatin, and zeatin riboside. Senescence was associated principally with a reduction in ability to form 7-glucosylzeatin, enhanced metabolism to adenine derivatives, and an inability to form appreciable amounts of 3-glucosyl-BAP. A two-dimensional thin layer chromatography (TLC) system, based on adjoining layers of cellulose and silica gel, for separating zeatin metabolites is described. This does not completely separate zeatin and zeatin riboside from the corresponding dihydro-compounds. A reversed phase TLC method for achieving these separations is also reported.     

Zeatin-9-glucoside,a major endogenous cytokinin of Vinca rosea L. crown gall tissue

Cultured crown gall tissue of Vinca rosea L. was found to contain, in addition to the previously reported cytokinins zeatin, zeatin riboside, and the 0-glucosides of these two compounds, relatively high levels of zeatin-9-D-glucopyranoside. This is the first conclusive identification of an endogenous cytokinin 9-glucoside.Abbreviations GC gas chromatography - HPLC high-performance liquid chromatography - I.D. internal diameter - RFE rotary film evaporation - TLC thin layer chromatography - TMS trimethylsilyl - UV ultraviolet - Z zeatin - Z7G zeatin-7-glucoside - Z9G zeatin-9-glucoside - Z0G zeatin-0-glucoside - ZR zeatin riboside - ZR0G zeatin riboside-0-glucoside     

Regulators of cell division in plant tissues XXIX. The activities of cytokinin glucosides and alanine conjugates in cytokinin bioassays

In a number of cytokinin bioassays, the activities of the following compounds were compared: 3-, 7-, and 9-glucosides of 6-benzylaminopurine (BAP); 7- and 9-glucosides of zeatin; O-glucosides of zeatin, dihydrozeatin, and their ribosides; 9-alanine conjugates of zeatin, and BAP. The bioassays included the radish cotyledon, theAmaranthus betacyanin, the oat leaf senescence, and the tobacco pith callus. Cytokinin activity was markedly reduced by 7- and 9-glucosylation in nearly all bioassays, but 3-glucosylation of BAP and O-glucosylation of the zeatin sidechain usually had little effect on activity. However, there were two notable exceptions to this generalization: the activity of O-glucosylzeatin markedly exceeded that of zeatin in the oat leaf senescence assay; 9-glucosyl-BAP and free BAP were similarly active in retarding the senescence of radish leaf discs. The 9-alanine conjugate of zeatin (lupinic acid) and of BAP were markedly less active than zeatin and BAP, respectively, in all bioassays, but the responses evoked by these conjugates at high concentrations in theAmaranthus bioassay approached those caused by the corresponding base. The activities of several new compounds related to the alanine conjugate of BAP were also assessed. To serve as a guide in the selection of the most suitable bioassay for detection of the above-mentioned cytokinin conjugates, the lowest detectable amounts in selected bioassays have been compared.     

Mass spectrometric analysis of cytokinins in plant tissues : v. Identification of the cytokinin complex of datura innoxia crown gall tissue

The cytokinin complex of Datura innoxia Mill. crown gall tissue was purified by ion exchange, Sephadex LH-20 chromatography and reversed-phase high performance liquid chromatography. By gas chromatography-mass spectrometry using ²H-labeled compounds, the following cytokinins were identified in the basic fraction eluting from a cation exchange column: zeatin, zeatin riboside, dihydrozeatin, dihydrozeatin riboside, their corresponding O-glucosides, 7- and 9-glucosides of zeatin, 9-glucoside of dihydrozeatin, isopentenyladenine, and isopentenyladenosine. Zeatin riboside 5′-monophosphate was the major cytokinin nucleotide in the tissue. In addition, dihydrozeatin riboside and isopentenyladenosine were identified in the nucleotide fraction following enymic degradation.     

Biological activity of cytokinins derived from Ortho- and Meta-Hydroxybenzyladenine

To determine the structure-activity relationships of natural aromatic cytokinins, the activity of 6-benzylaminopurine (BAP) and its hydroxylated derivatives was compared in three bioassays based on stimulation of tobacco callus growth, retention of chlorophyll in excised wheat leaves, and dark induction of betacyanin synthesis in Amaranthus cotyledons. The aromatic cytokinins 6-(2-hydroxybenzylamino)purine (ortho-topolin) and 6-(3-hydroxybenzylamino)purine (meta-topolin), their 9-ribosides and 9-glucosides, were synthesized by the condensation of 6-chloropurine and its 9-glycosides with the appropriate hydroxybenzylamine. The activity of free bases, 9-ribosides and 9-glucosides was compared with that of BAP, trans-zeatin and their 9-glycosides. Hydroxylation of the benzyl ring in the meta position increased the activity of BAP and its riboside in tobacco callus and chlorophyll retention bioassays, whereas ortho-hydroxylation decreased the activity. In contrast, in the Amaranthus bioassay meta-hydroxylation of BAP substantially decreased its activity. Ribosylation at position 9 had no significant effect on the activity of zeatin, BAP and both topolins. The activity of 9-glucosides of all cytokinins tested was near zero. The biological activity of meta-topolin and its riboside is comparable to that of the most active isoprenoid cytokinin, zeatin, in tobacco callus growth and senescence bioassays. The results establish the existence of a family of endogenous aromatic cytokinins centered around the highly active compound, meta-topolin. We also report here an improved chlorophyll retention bioassay based on incubation of 2.5 cm long detached wheat leaf segments in microtiter plate wells containing 150 µl of cytokinin solution. The consumption of cytokinin to be tested is 0.1 µmol per assay only. The amount as small as 1.5 pmol of substance can be estimated using this biotest.     

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     

Endogenous cytokinins in vegetative shoots of peas

In G2 peas senescence only takes place in long days. In order to determine the role of cytokinins in this process the endogenous cytokinins from vegetative shoots of G2 peas were characterized using gas chromatography-mass spectroscopy following purification by HPLC. Cytokinins were extracted and purified with and without the addition of ¹⁵N labelled internal standards of several cytokinins to estimate cytokin content by isotope dilution in the mass spectra. Samples without internal standards were bioassayed after HPLC. Bioassays showed the presence of zeatin, zeatin riboside and zeatin-0-glucoside. The presence of zeatin was confirmed by its mass spectrum of its permethylated derivative. Tentative identification of zeatin riboside, zeatin-0-glucoside, dihydrozeatin, and dihydrozeatin-0-glucoside was obtained by the coincidence of the major ion for the permethylated natural and ¹⁵N labelled internal standards on GC-MS, and the similar coincidence of ions for permethylated zeatin riboside-0-glucoside by direct probe MS. There was no indication of the presence of significant quantities of zeatin-7-glucoside or zeatin-9-glucoside. The amounts in the tissue ranged from 200–1000 ng/kg fresh weight for each cytokinin and about 2–4 g/kg fresh weight for total cytokinins. There was no apparent difference in the levels in mature but pre-senescent shoots grown in long days and short days indicating that apical senesecence in G2 peas does not appear to be induced by a decline in cytokinin level in the shoots.Cytokinin abbreviations CK Cytokinin - Z trans zeatin - [9R]Z t-zeatin riboside - [9R-5P] Z t-zeatin riboside-5-monophosphate - (OG)Z t-zeatin-0-glucoside - (OG)[9R]Z t-zeatin riboside-0-glucoside - [7Z]G t-zeatin-7-glucoside - [9G]Z t-zeatin-9-glucoside - (diH)Z dihydrozeatin - (diH)[9R]Z dihydrozeatin riboside - iP N6(²-isopentenyl) adenine - [9R]iP N6(²-isopentenyl) adenosine Work performed while PJD was on leave at the University College of Wales at Aberystwyth.     

The stability and biological activity of cytokinin metabolites in soybean callus tissue

The activity, uptake and metabolism of cytokinin metabolites was determined in soybean (Glycine max (L.) Merr.) callus tissue. The following activity sequence was established: zeatin riboside (ZR)>zeatin (Z)>O-glucosides of Z, ZR and their dihydro derivatives>lupinic acid (an alanine conjugate of Z)>7- and 9-glucosides of Z which were almost inactive. The 7- and 9-glucosides and lupinic acid were taken up very slowly by the callus tissue and showed great metabolic stability, but some degradation to 7-glucosyladenine, 9-glucosyladenine and the 9-alanine conjugate of adenine occurred. Compared with its aglycone, O-glucosyl-ZR exhibited slow uptake and greatly enhanced stability but gas chromatographic-mass spectrometric analysis showed that appreciable amounts were hydrolyzed to ZR in the tissue. Both ZR and O-glucosyl-ZR were metabolised extensively, with adenine, adenosine, and adenine nucleotide(s) as the major metabolites. A diversity of minor metabolites of ZR were identified, including O-glucosides, lupinic acid and dihydrolupinic acid. The metabolism of ZR was suppressed by 3-isobutyl-1-methylxanthine. When compared with the soybean callus line normally used for cytokinin bioassays (cv. Acme, cotyledonary callus), related callus lines exhibited greatly differing growth responses to cytokinin: however, these were not reflected in marked differences in metabolism.Abbreviations GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - LA lupinic acid - OGZR O--D-glucopyranosylzeatin riboside - TLC thin-layer chromatography - IMX 3-isobutyl-1-methylxanthine - Z zeatin - ZR zeatin riboside     

Influence of White Clover Mosaic Potexvirus Infection on the Endogenous Cytokinin Content of Bean

The cytokinin content in the primary leaves of bean (Phaseolus vulgaris) was monitored for 10 d after inoculation with white clover mosaic potexvirus. The cytokinins were isolated, purified, separated by high-performance liquid chromatography, and quantified by radioimmunoassay. The cytokinins detected at the time of inoculation (d 0) were: (a) the free bases, zeatin (Z), dihydrozeatin (DZ), and isopentenyladenine; (b) the riboside, DZ riboside (DZR); (c) the O-glucosides of DZ, DZR, and Z riboside; (d) the nucleotides, Z riboside-5′-monophosphate and isopentenyladenosine-5′-monophosphate; and (e) trace amounts of Z-9-glucoside and DZ-9-glucoside. During the 10 d after inoculation with white clover mosaic potexvirus, marked quantitative changes in this cytokinin profile were observed. The concentration of the free bases and DZR decreased, accompanied by an increase in the 9-glucosides and the nucleotides. Virus titer increased rapidly 3 d after inoculation, attaining a maximum level at d 5. This increase coincided with the increases in the 9-glucosides and the nucleotides. We propose that the decline in the cytokinin free bases and riboside may allow the increase of virus titer in bean and lead to the senescence of infected leaves.     

Regulators of cell division in plant tissues

The cytokinins in certain fractions prepared from extracts of immature sweet-corn (Zea mays L.) kernels using polystyrene ion-exchange resins have been further investigated. Cytokinins active in the radish cotyledon bioassay were purified from these fractions and identified as 9--D-glucopyranosylzeatin, 9--D-glucopyranosyldihydrozeatin, O--D-glucopyranosylzeatin. and O--D-glucopyranosyl-9--D-ribofuranosylzeatin. In addition, compounds which resemble zeatin and its glycosides in chromatographic behaviour and in ultraviolet absorption characteristics were purified from extracts of the same material by high-performance liquid chromatography. In addition to zeatin and zeatin riboside, the following compounds were identified unambiguously: O--D-glucopyranosyl-9--D-ribofuranosyldihydrozeatin, O--D-glucopyranosyldihydrozeatin, and hihydrozeatin riboside. A further compound was tentatively identified as O--D-glucopyranosylzeatin, and at least two unidentified compounds appeared to be new derivatives of zeatin. In identifying the above compounds, chemical-ionization mass spectrometry proved to be an invaluable complementary technique, yielding spectra showing intense protonated-molecular-ion peaks and also prominent structure-related fragmentation that was either not evident or very minor in the electron-impact spectra. An assessment of the relative importance of the various possible mechanisms for cytokinin modification and inactivation in mature sweet-corn kernels was made by supplying [³H]zeatin and [³H]zeatin riboside to such kernels after excision. The principal metabolites of zeatin were adenine nucleotides, adenosine and adenine, while little of the metabolite radioactivity was attributable to known O-glucosides. Adenine nucleotides and adenine were the principal metabolites of zeatin riboside, while lesser metabolites were identified as adenosine, dihydrozeatin, and the O-glucosides of dihydrozeatin and dihydrozeatin riboside. Side-chain cleavage, rather than side-chain modification, appears to be the dominant form of cytokinin metabolism in mature sweet-corn kernels.Abbreviations CI-MS chemical-ionization mass spectrum - EIMS electron-impact mass spectrum - GC-MS combined gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - M⁺ molecular ion - MH⁺ protonated molecular ion - TLC thin-layer chromatography - TMS trimethylsilyl - UV ultraviolet XXVII=Letham et al. (1979)     

Direct and efficient plant regeneration from leaf explants of Solanum tuberosum l. cv. Bintje

Summary A two-step procedure was used for plant regeneration from in vitro grown leaf strips (2–3 mm wide) of cv. Bintje. Step I medium was designed with 2,4-dichlorophenoxycetic acid (2,4-D) at 0.0 or 9.0 M, in combination with 2.28 M kinetin (K), benzyl adenine (BA), zeatin (Z) or zeatin riboside (ZR). Step II media were 2,4-D-free media containing 5.78 M gibberellic acid (GA3) and growth regulators similar to those of step I media. Leaf explants cultured in medium I containing zeatin riboside or zeatin for 6 days and then subcultured in medium II containing zeatin riboside produced numerous shoots without callus formation. Zeatin riboside containing step I and II media caused shoot regeneration in a high number (97.5±2.2) of explants. Approximately, 33.7±8.4 shoots were regenerated from each leaf explant.Abbreviations BA benzyladenine - Z zeatin - ZR zeatin riboside (trans isomer) - 2,4-D 2,4-dichlorophenoxyacetic acid     

Chicken egg yolk antibodies for cytokinin analysis

The production, isolation, and purification of specific chicken immunoglobulins (Igs) against three main groups of naturally occurring cytokinins are reported. The specific Igs directed against, respectively, zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine are extracted from the egg yolk and used in radioimmunoassays that allow the quantification in parallel of pmol of the cytokinins in plant extracts. As little as 50 fmol of zeatin riboside, 20 fmol of isopentenyladenosine, and 40 fmol of dihydrozeatin riboside can be detected. The levels of cytokinins measured in the radio-immunoassay correlate well with physicochemical analysis methods such as high performance liquid chromatography (HPLC) with UV spectrum detection and HPLC-coupled mass spectrometric detection. Cross-reactivity studies indicate that the assay is not affected by most of the structurally related compounds. The respective antibody preparations recognized zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine and the corresponding free bases. The results obtained when analyzing crude plant extracts are expressed as zeatin riboside equivalents, dihydrozeatin riboside equivalents, and isopentenyladenosine equivalents.Abbreviations B binding activity - B ₀ maximal binding - B ₁ unspecific binding - GC gas chromatography - HPLC high performance liquid chromatography - LC-MS HPLC-coupled mass spectrometry - MOPS 4-morpholinepropanesulfonic acid - RIA radioimmunoassay - TBS Tris-buffered saline - (diH)Z dihydrozeatin - (diH) [9R]Z dihydrozeatin riboside - iP isopentenyladenine - [9R]iP isopentenyladenosine - Z zeatin - [9R]Z zeatin riboside - [9G]iP isopentenyladenine-9-glucoside - [9R-5P]iP isopentenyladenosine-5-monophosphate     

Methyl jasmonate down-regulates endogenous cytokinin levels in cotyledons of Cucurbita pepo (zucchini) seedlings

Endogenous cytokinins (CK) were measured in Cucurbita pepo L. (zucchini) cotyledons after spraying 7-day-old seedlings with methyl jasmonate (MeJA) to examine whether the senescence-promoting action of MeJA is associated with changes in metabolic regulation of CK levels. MeJA promoted senescence as estimated by the loss of chlorophyll and injury to chloroplast ultrastructure. This was already detected one day after treatment. The contents of physiologically active CKs determined by high-performance liquid chromatography-mass spectrometry (CK bases: trans -zeatin, dihydrozeatin and isopentenyladenine and their ribosides), especially trans -zeatin and its riboside declined considerably. CK nucleotides and physiologically inactive CK 7- and 9-glucosides were also markedly decreased whereas the content of storage CK O -glucosides as well as cis -isomers of zeatin declined only slightly. Chlorophyll content and the normal chloroplast structure recovered 5 days after the MeJa treatment. The process of photosynthetic apparatus recovery correlated with an increased concentration of physiologically active CKs, especially Z. In contrast, the content of CK nucleotides and storage CK O -glucosides decreased further. We suggest that the ability of MeJA to promote some aspects of senescence in intact zucchini cotyledons is at least partially due to down-regulation of endogenous CK levels and their interconversion between active and inactive forms.     

Mass-spectrometric quantitation of cytokinins in tobacco crown-gall tumours induced by mutated octopine Ti plasmids of Agrobacterium tumefaciens

The levels of the major cytokinins, zeatin, zeatin riboside, zeatin riboside-5-monophosphate and zeatin-7-glucoside were measured in tobacco (Nicotiana tabacum L.) crown-gall tissues carrying insertion and deletion mutations in the T-DNA. Measurements were made by combined gas chromatography-mass spectrometry using selected ion monitoring with ¹⁵N- and ²H-labelled internal standards. The results demonstrate that, relative to wild-type tumour tissue, cytokinin levels are considerably elevated in tissues lacking functional T-DNA auxin-biosynthetic genes. From a detailed analysis of the major cytokinin metabolites it is concluded that a reduction in the extent of cytokinin degradation via N⁶-side-chain cleavage is an important factor leading to increased cytokinin levels in these tissues.Abbreviations IAA indole-3-acetic acid - SIM selected ion monitoring - Z zeatin - [7G]Z zeatin-7-glucoside - [9R]Z zeatin-9-riboside - [9R-5P]Z zeatin riboside-5-monophosphate     

The effect of aluminum on cytokinins in the mycelia of Amanita muscaria

High performance liquid chromatography analysis of immunoaffinity-purified extracts of mycelia of Amanita muscaria, and the Amaranthus bioassay of the eluted fractions, revealed the following seven cytokinins: zeatin, zeatin riboside, zeatin N-9-glucoside, dihydrozeatin, dihydrozeatin riboside, isopentenyl adenine, and isopentenyl adenosine. The decreased growth of aluminum-treated mycelia correlated with a 35% decrease in the total amount of the cytokinins. Among individual cytokinins, zeatin was the most affected, exhibiting a reduction of about 90%. The results are compared with previous investigations of aluminum effects on cytokinins in the mycelia of Lactarius piperatus, whose growth is stimulated by aluminum.Abbreviations ZR zeatin riboside - iPA isopentenyl adenosine - Z zeatin - DHZ dihydrozeatin - iP isopentenyl adenine - DHZR dihydrozeatin riboside - Z-9G zeatin N-9-glucoside - iP-9G isopentenyl N-9-glucoside - HPLC high performance liquid chromatography - DHZRMP dihydrozeatin riboside monophosphate - ZRMP zeatin riboside monophosphate     

Regulators of cell division in plant tissues

[³H]zeatin was supplied through the transpiration stream to de-rooted lupin (Lupinus angustifolius L.) seedlings. The following previously known metabolites were identified chromatographically: 5-phosphates of zeatin riboside and dihydrozeatin riboside, adenosine-5-phosphate, zeatin riboside, zeatin-7-glucopyranoside, zeatin-9-glucopyranoside, adenine, adenosine and dihydrozeatin. Five new metabolites were purified; four of these contain an intact zeatin moiety. Two were identified unequivocally, one as l--[6-(4-hydroxy-3-methylbut-trans-2-enylamino)-purin-9-yl]alanine, a metabolite now termed lupinic acid, and the second as O--d-glucopyranosylzeatin. These two compounds were the major metabolites formed when zeatin solution (100 M) was supplied to the de-rooted seedlings. The radioactivity in the xylem sap of intact seedlings, supplied with [³H]zeatin via the roots, was largely due to zeatin, dihydrozeatin and zeatin riboside. When [³H]zeatin (5 M) was supplied via the transpiration stream to de-rooted Lupinus luteus L. seedlings, the principal metabolite in the lamina was adenosine, while in the stem nucleotides of zeatin and adenine were the dominant metabolites. O-Glucosylzeatin and lupinic acid were also detected as metabolites. The level of the latter varied greatly in the tissues of the shoot, and was greatest in the lower region of the stem and in the expanding lamina. Minor metabolites also detected chromatographically were: (a) dihydrolupinic acid, (b) a partially characterized metabolite which appears to be a 9-substituted adenine (also formed in L. angustifolius), (c) glucosides of zeatin riboside and/or dihydrozeatin riboside, and (d) O-glucosyldihydrozeatin. While lupinic acid supplied exogenously to L. luteus leaves underwent little metabolism, chromatographic studies indicated that O-glucosylzeatin was converted to its riboside, the principal metabolite formed, and also to adenosine, zeatin and dihydrozeatin. A thinlayer chromatography procedure for separating zeatin, dihydrozeatin, zeatin riboside and dihydrozeatin riboside is described.Abbreviations Me₃Si trimethylsilyl - TLC thin-layer chromatography - UV ultraviolet XXIV=Gordon et al., 1975     

Endogenous isoprenoid and aromatic cytokinins in different plant parts of Cocos nucifera (L.)

The present study reports the analyses of both isoprenoid and aromatic cytokinins in the coconut palm by combined high performance liquid chromatography and group specific enzyme immunoassays (HPLC-ELISA). The results showed that the isoprenoid cytokinins were several fold more abundant than the aromatic cytokinins in each of the plant parts analysed: immature inflorescence, shoot apical meristem (SAM), spear leaf and embryo. Within the isoprenoid cytokinins, the most abundant ones by type were the zeatin- (Z-), the isopentenyladenine- (iP-) and the dihydrozeatin- (DHZ-) type in decreasing order for most plant parts studied, and individually, zeatin riboside (ZR) or zeatin riboside-5-monophosphate (ZR5P) depending on the part. In the case of the iP-type cytokinins, the results showed that its 9-glucoside was the most abundant one in most parts. The isoprenoid cytokinin profiles in coconut showed a predominant pattern of 9-conjugation as a major metabolism route for these cytokinins. Analyses also showed the occurrence of the aromatic cytokinin 6-benzylaminopurine (BAP) and its riboside (BAPR), 9-glucoside (BAP9G), and nucleotide (BAPR5P). Their presence in coconut palm was unequivocally identified after permethylation by gas chromatography-mass spectrometry. They were more concentrated in the embryo and in the immature inflorescence than in the other two parts studied, however their concentration in each part was several times lower than that of isoprenoid cytokinins. All four were detected in each of the parts studied. The most abundant ones were BAPR and BAP9G in immature inflorescence; and BAPR in all of the other parts. When all cytokinins analysed are considered, differences between the plant parts studied were found. The zygotic embryos showed the highest content, double that in immature inflorescence, and five times more that in spear leaf and SAM. These differences are even greater when individual cytokinins are compared.     

Involvement of cytokinins in the germination of chick-pea seeds

Eight cytokinins detected in germinated chick-pea (Cicer arietinum L. var. Castellana) seeds were first present in the embryonic axes but appeared in the cotyledons after 12h of germination. The cytokinins detected in the cotyledons originate in the embryonic axes, but no passage of these substances from the cotyledons to the axes was detected, except when the seeds were treated with red light.It is concluded that the role played by the embryonic axis in mobilizating the main reserves of the cotyledons is mainly effected through these cytokinins. Both natural and synthetic cytokinins exert an important regulatory role in the hydrolysis of reserve proteins and calcium could be involved as an intermediate.Abbreviations BA benzyladenine - cot. cotyledon - (diH)Z dihydrozeatin - (diH)ZR dihydrozeatin riboside - GZR glycosyl zeatin riboside - 2iP 277-1 - iPA 277-2 riboside - Kin kinetin - Z zeatin - ZG zeatin glucoside - ZR zeatin riboside     

Isolation of two cytokinin metabolites from the rhizosphere of Norway spruce seedlings (Picea abies L. Karst.)

Roots of young Norway spruce seedlings were incubated under hydroculture conditions in a synthetic nutrient medium containing either ³H-isopentenyladenosine, isopentenyladenosine or zeatin riboside. When feeding with ³H-isopentenyladenosine a new radiaolabelled metabolite was found in the feeding solution as well as in root extracts. Isopentenyladenosine and zeatin riboside were metabolised and for both compounds an unknown metabolite was detected in the feeding solution. The metabolites were purified by solid phase extraction, HPLC and partially characterised. A major characteristic of the metabolites is their reactivity in the presence of NH₄OH, which results in the formation of the cytokinin bases isopentenyladenine or zeatin, respectively. UV-spectra and the chemical characteristics indicate that the new metabolites are closely related. The GC-MS analysis revealed, that the metabolites are true derivatives of isopentenyladenine and zeatin. The biogenesis of the new metabolites is discussed with regard to plant microbial interactions.Abbreviations Ck(s) = cytokinin(s) - GC-MS = gas chromatography-mass spectrometry - iP = isopentenyladenine - [9R]iP = isopentenyladenosine - [9G]iP = isopentenyladenine-9-glucoside - [9R-MP]iP = isopentenyladenosine-5-monophosphate - Z = trans-zeatin - [9R]Z = trans-zeatin riboside