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     

Cytokinin metabolism in Phaseolus vulgaris L.

The major cytokinins in stems of decapitated, disbudded bean plants have been identified by enzymic degradation, Sephadex LH20 and reversed phase high performance liquid chromatography, and by combined gas chromatography-mass spectrometry as 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-ribofuranosylpurine (zeatin riboside), 6-(4-hydroxy-3-methylbutylamino)-9--D-ribofuranosylpurine (dihydrozeatin riboside), and the 5-phosphates of these compounds (zeatin ribotide and dihydrozeatin ribotide). Minor cytokinins in this tissue were tentatively identified as dihydrozeatin-O--D-glucoside and zeatin ribotide-O--D-glucoside. [8-¹⁴C-]Dihydrozeatin appeared to be rapidly metabolized to dihydrozeatin ribotide when supplied to segments of stems from decapitated plants. These results are discussed in relation to the metabolism and distribution of cytokinins in the whole plant.Abbreviations TEAB triethyl ammonium bicarbonate - UV ultra-violet - GCMS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - TMS trimethyl silyl     

Dihydrozeatin riboside,a minor cytokinin from the leaves of Phaseolus vulgaris L.

Dihydrozeatin riboside has been identified in the leaves of decapitated bean plants by Sephadex LH20 chromatography and combined gas chromatography-mass spectrometry. The relationship between the cytokinins isolated and identified from this system and those previously reported in Phaseolus is discussed.Abbreviations DHZ dihydrozeatin - DHZOG dihydrozeatin-O--D-glucoside - DHZR dihydrozeatin riboside - GCMS combined gas chromatography-mass spectrometry - GLC gas-liquid chromatography - TIC total ion current - TMS trimethylsilyl - Z zeatin - ZR zeatin riboside     

Morphology of potato (Solanum tuberosum L. cv. Sante) stem node cultures in relation to the level of endogenous cytokinins

Stem node culture of the potato (Solanum tuberosum) cv. Sante was used to examine the phenotypical alterations due to different levels of endogenous cytokinins. The altered phenotype, which dramatically deviates from the control phenotype, was induced after treatment of plantlets with 1 m jasmonic acid. Plantlets grown on the medium supplemented with jasmonic acid were taller, with well developed root systems, expanded leaves, thickened stems, and they showed hyperhydric symptoms. Their cytokinin content was about half that of the control plantlets. Morphologic characteristics corresponding to transgenic plants that overproduce cytokinins, including release of axillary buds and inhibited rooting, correlated with the high cytokinin levels in control plants.Abbreviations JA jasmonic acid - Z trans-zeatin - ZR trans-zeatin riboside - ZRMP zeatin riboside 5-monophosphate - Z-9-G trans-zeatin N-9-glucoside - DHZ dihydrozeatin - DHZR dihydrozeatin riboside - DHZRMP dihydrozeatin riboside 5-monophosphate - DHZ-9-G dihydrozeatin 9-glucoside - iP iso-pentenyladenine - iPA iso-pentenyladenosine - iP-9-G iso-pentenyladenine 9-glucoside - HPLC high performance liquid chromatography     

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     

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.     

Postharvest changes in endogenous cytokinins and cytokinin efficacy in potato tubers in relation to bud endodormancy

Using an indirect enzyme‐linked immunosorbent assay (ELISA), the effects of postharvest storage duration and temperature on endogenous cytokinins in potato ( Solanum tuberosum L. cv. Russet Burbank) tuber apical bud tissues in relation to endodormancy status were determined. Following fractionation by HPLC, a total of eight cytokinins were detected and these were: zeatin riboside‐5'‐monophosphate (ZRMP), zeatin‐ O ‐glucoside (ZOG), zeatin (Z), zeatin riboside (ZR), isopentenyl adenosine‐5'‐monophosphate (IPMP), isopentenyl adenine‐9‐glucoside (IP‐9‐G), isopentenyl adenine (IP) and isopentenyl adenosine (IPA). Regardless of postharvest storage temperature or endodormancy status, IP‐9‐G was the most abundant cytokinin detected while ZRMP and ZOG were the least abundant ones. In tubers preincubated at a growth‐permissive temperature (20°C) prior to extraction, the loss of endodormancy was preceded by significant increases in the endogenous levels of Z, ZR, IPMP and IP‐9‐G. When stored continuously at a growth‐inhibiting temperature (3°C), significant increases in ZR, IP‐9‐G and IP + IPA were observed. The total content of cytokinins increased by over 7‐fold during postharvest storage and this increase was a result of de novo biosynthesis. Dose‐response studies using IPA and ZR demonstrated a time‐dependent increase in apparent cytokinin sensitivity during postharvest storage. With the exception of IP‐9‐G, injection of any of these endogenous cytokinins resulted in the rapid and complete termination of tuber endodormancy. The significance of these results with respect to endodormancy regulation and the possible mechanisms controlling cytokinin levels in potato tubers are discussed.     

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.     

Dormancy-related changes in cytokinin efficacy and metabolism in potato tubers during postharvest storage

The metabolism of [³H]-zeatin (Z) and[³H]-isopentenyladenosine (IPA) in potato tubers was examined inrelation to changes in cytokinin efficacy during postharvest storage anddormancy progression. Exogenous radiolabeled cytokinins were rapidlymetabolizedby dormant and nondormant tubers. Following injection, [³H]-Z wasmetabolized to zeatin riboside, adenine derivatives andzeatin-riboside-5-monophosphate. Four hours after injection, less than60% of the recovered radioactivity was associated with unmetabolized[³H]-Z. [³H]-IPA was also rapidly metabolized to severalmetabolites including: IPA-5-monophosphate, adenine derivatives andzeatin riboside. Four hours after injection, less than 50% of therecovered radioactivity was associated with [³H]-IPA. Cytokininsensitivity was assessed by determining the effects of exogenous Z or IPA ontuber sprouting. Immediately after harvest and during the initial period ofstorage, tubers were dormant and exogenous Z or IPA were completely ineffectivein breaking tuber dormancy. Thereafter, dormant tubers exhibited a gradualincrease in sensitivity to both cytokinins. Cytokinin sensitivity continued toincrease as postharvest storage was extended and dormancy weakened. The lengthof postharvest storage (hence dormancy status) had no apparent effects on themetabolism of either cytokinin. Neither the rate of metabolism nor the natureofmetabolites detected was affected by the length of postharvest storage. Theseresults suggest that changes in cytokinin efficacy in dormant potato tubersduring postharvest storage are not the result of differential catabolism butrather are due to other cellular processes such as hormone perception and/orsignal transduction.     

Changes in Concentrations of Cytokinins (CKs) in Root and Axillary Bud Tissue of Miniature Rose Suggest that Local CK Biosynthesis and Zeatin-Type CKs Play Important Roles in Axillary Bud Growth

Involvement of cytokinins (CKs) in axillary bud growth of miniature rose was studied. Variation in root formation and axillary bud growth was induced by two indole 3-butyric acid (IBA) pretreatments in two cutting sizes. At six physiological developmental stages around the onset of axillary bud growth, concentrations of CKs were determined in both root and axillary bud tissue by liquid chromatography combined with electrospray tandem mass spectrometry (LC-ESP-MS/MS). Chronological early onset of axillary bud growth occurred in long cuttings pretreated at low IBA concentration, whereas physiological early root formation was associated with long cuttings and high IBA concentration. The CKs zeatin (Z), isopentenyl adenine (iP), zeatin riboside (ZR), dihydrozeatin riboside (DHZR), isopentenyl adenosine (iPA), zeatin O-glucoside (ZOG), zeatin riboside O-glucoside (ZROG), zeatin riboside 5′-monophosphate (ZRMP), and isopentenyl adenosine 5′-monophosphate (iPAMP) were detected. Concentrations of CKs in axillary bud tissue far exceeded those in root tissue. Indole 3-butyric acid pretreatment influenced the concentration of CKs in axillary bud tissue more than did cutting size, whereas pretreatments only slightly affected CKs in root tissue. The dominant CKs found were iPAMP and ZR. An early and large increase in iPAMP indicated rapid CK biosynthesis in rootless cuttings, suggesting that green parts, including the axillary bud, can synthesize CKs. At the onset of axillary bud growth an increase in concentration of Z, ZR, ZRMP, ZOG, and ZROG was largely coincident with a decrease in iPAMP, iPA, iP, and DHZR. After the onset of axillary bud growth, CK content largely decreased. These results strongly indicate a positive role for CKs in axillary bud growth, and presumably ZRMP, ZR, and Z are active in miniature rose.     

Occurrence of aromatic cytokinins in oil palm (Elaeis guineensis Jacq.)

The natural occurrence of 6-benzylaminopurine, 6-(2-hydroxybenzylamino)purine (ortho-topolin), 6-(3-hydroxybenzylamino)purine (meta-topolin), their ribosides and 9-glucosides is reported using specific antibodies to these groups of compounds in high performance liquid chromatography/enzyme-linked immunosorbent assay (HPLC/ELISA). Compounds were identified by their retention times and differential cross-reactivities with six antisera in analyses carried out in two laboratories using different HPLC gradient systems. Identities were confirmed by immunoaffinity purification followed by HPLC with on-line UV spectrum analysis. Further confirmation of the occurrence of ortho-topolin riboside and isopentenyladenine-9-glucoside was obtained from gas chromatography-mass spectrometry analysis of permethylated HPLC fractions of an extract of oil palm tissues. The aromatic cytokinins, and in particular, ortho-topolin riboside, were found in a variety of oil palm tissues at concentrations exceeding those of the isoprenoid cytokinins, zeatin, isopentenyladenine, dihydrozeatin, and their ribosides. The 9-glucosides of isopentenyladenine and zeatin were more abundant than those of the aromatic types. The cross-reactivity of benzyladenine compounds with antibodies to isopentenyladosine is discussed in relation to the interpretation of ELISA data.Abbreviations BA N ⁶-benzyladenine - HPLC high performance liquid chromatography - ELISA enzyme-linked immunosorbent assay - GC-MS gas-chromatography-mass spectrometry - mT meta-topolin - oT ortho-topolin - TEAA triethylammonium acetate - IAC immunoaffinity chromatography - SPD spectral photodiode     

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)     

Properties of a high-affinity cytokinin-binding protein from wheat germ

A soluble protein that interacts with a range of cytokinins was extensively purified from wheat (Triticum aestivum L.) germ. This protein has a K _d for kinetin of 2×10^-7 M. The binding of kinetin to the protein is inhibited by low concentrations of synthetic and naturally-occurring cytokinins including N⁶-benzyladenine, N⁶-benzyladenosine, kinetin riboside, N⁶-dimethylallyladenine, N⁶-dimethylallyladenosine, zeatin, zeatin riboside, N⁶-dimethyladenine and N⁶-dimethyladenosine. Adenine, adenosine and several non-N⁶-substituted adenine derivatives were ineffective as inhibitors of kinetin binding. While N⁶-butyryl-3,5-cyclic AMP, N⁶,2-O-dibutyryl-3,5-cyclic AMP and 2,3-cyclic AMP inhibited binding of kinetin to the protein, 3,5-cyclic AMP was ineffective. The kinetin-binding protein is heat-labile and pronase-sensitive. Kinetin-binding activity exactly co-chromatographs with a single peak of carbohydrate and protein on gel-filtration and is displaced from concanavalin A-Sepharose 4B by -methylglucoside. On gel filtration, the kinetin-binding protein behaves as a soluble protein with an apparent molecular weight of 180,000 daltons.     

Uptake and metabolism of benzyladenine during shoot organogenesis in Petunia leaf explants

Benzyladenine (BAP) uptake and metabolism were characterized during the key stages of shoot organogenesis in leaf explants of Petunia MD1. Using leaf explant transfer experiments, it was shown that exposure to 2.2 M BAP for 6, 8 or 10 days induced shoot formation on 27, 80 and 100% of the explants respectively, with a concomitant increase in the number of shoots per explant. BAP uptake and metabolism were characterized in leaf explants after 1, 3, 6 or 10 days exposure to [³H]BAP or 10 days exposure plus an additional 2 days on basal medium (10+2). BAP and 9--D-ribofuranosyl-BAP ([9R]BAP) were detected at days 1 and 3 only. Therefore, the BAP free base was not detectable during the shoot induction period between days 6 and 10, as defined by leaf transfer experiments. The BAP ribotide pool was largest on day 1 and decreased to day 10+2. It is possible that the BAP ribotide pool provided either the active cytokinin itself or acted as a short-term storage form for the active cytokinin in petunia shoot organogenesis. Other metabolites detected in petunia leaf tissue included 7--D-glucopyranosyl-BAP ([7G]BAP), 9--D-glucopyranosyl-BAP ([9G]BAP) and an unidentified metabolite C.Abbreviations BAP benzyladenine - [7G]BAP 7--D-glucopyranosyl-BAP - [9G]BAP 9--D-glucopyranosyl-BAP - [9R]BAP 9--D-ribofuranosyl-BAP - [9R-5P]BAP 5-monophosphate of [9R]BAP - [9R-5PP]BAP 5-diphosphate of [9R]BAP - [9R-5PPP]BAP 5-triphosphate of [9R]BAP - TEA Triethylamine This research was supported in part by NSF Grant DCB-8917378 to J.D.C. and USDA-CRGO Grant 89-37261-4791 to T.J.C.     

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     

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     

Cytokinins in transfer RNA of normal and crown-gall tissue of Vinca rosea

cis-Zeatin riboside was identified in transfer-RNA hydrolysates from both normal and crown-gall tissue of Vinca rosea L. The trans-isomer was associated exclusively with the crowngall transfer-RNA. The importance of these observations is discussed in relation to biosynthesis of free cytokinins.Abbreviations GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - TLC thin-layer chromatography - TMSi trimethylsilyl - tRNA transfer RNA - ZR zeatin riboside     

A tracheid-differentiation factor from pine needles

Exogenous indol-3yl-acetic acid (IAA), alone and together with several cytokinins, was ineffective in promoting the complete differentiation into tracheids of cambial derivatives of Pinus contorta Dougl.; IAA alone promoted cambial cell division and primary-wall growth in cambial derivatives throughout the stem's length. In contrast, a single pair of needles on a stem cutting in light promoted neither cambial cell division nor primary-wall growth in cambial derivatives but did promote complete differentiation of cambial cells into tracheids; tracheids differentiated only near the junction of the foliated short shoot with the stem. Clear inter-and intracellular differences in the extent of tracheid differentiation occurred in response to a single needle pair and have suggested the hypothesis that a specific tracheid-differentiation factor regulates the differentiation of cells into proto-, meta-, or secondary-xylem tracheary elements through an interaction with IAA.Abbreviations IAA indol-3yl acetic acid - K kinetin - BAP 6-benzylaminopurine - Z zeatin - ZR zeatin riboside - FAA formalin: acetic acid: ethanol: water (10:5:50:35, by vol.)     

Identification of the cytokinins in red pine seedlings

Zeatin-9-riboside was identified in shoots and roots of Pinus resinosa by GC-MS analysis of its permethyl derivative. Based on their chromatographic properties on Sephadex LH-20 and C₁₈ HPLC, and their susceptibility to enzymatic degradation, several other cytokinins have been tentatively identified. The basic fraction of both the roots and shoots contained zeatin, whereas the shoots contained dihydrozeatin-O-glucoside and the roots contained zeatin-O-glucoside. Zeatin-9-riboside monophosphate, isopentenyladenosine monophosphate ([9R-5P]iP) and glucosyl phosphate derivatives were detected in the acidic fractions from both roots and shoots. There were equivalent amounts of [9R-5P]iP in both roots and shoots. The presence of equivalent amounts of [9R-5P]iP in both the roots and shots suggests that cytokinin biosynthesis may be occurring in both locations.Abbreviations AMP adenosine-5-monophosphate - BAP benzylaminopurine - BSA bovine serum albumin - BuOH butan-1-ol - CK cytokinin - (diH)Z dihydrozeatin - (diH OG)Z dihydrozeatin-O-glucoside - (diH OG)[9R]Z dihydrozeatin-9-riboside-O-glucoside - DW dry weight - EtOH ethanol - FW fresh weight - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - [9R]iP isopentenyladenosine - [9R-5P]iP isopentenyladenosine monophosphate - MeOH methanol - PVP polyvinylpyrrolidone - RFE rotary film evaporation - TEAB triethyl ammonium bicarbonate - Z zeatin - [9R]Z zeatin-9-riboside - (OG)Z zeatin-O-glucoside - [7G]Z zeatin-7-glucoside - [9R-5P]Z zeatin-9-riboside monophosphate     

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