Plant growth regulators as putative physiological markers of developmental stage in Prunus persica

Prunus persica plants of different ages and statesof maturation were analysed to compare their phytohormonal status. Endogenouslevels of indole-3-acetic acid (IAA), abscisic acid (ABA), and severalcytokinins (Cks): zeatin (Z), zeatin riboside ([9R]Z), dihydrozeatin ((diH)Z),dihydrozeatin riboside ((diH)Z[9R]), isopentenyl adenine (iP) and isopentenyladenosine ([9R]iP), were measured in order to determine their possible use asphysiological indices of phase change and maturation. A decrease in Ck levels(Z, [9R]Z, (diH)Z, (diH)[9R]Z, and iP, [9RiP) was found from the embryonic tojuvenile stage as well as a decrease in the ratio of iP-type (iP and[9R]iP)/Z-type Cks paralleling the increase in tree age. ABA levels increasedduring maturation in Prunus persica and the ratio ofCks/IAA decreased with tree age. From our results, we propose that the balancesof Cks/IAA and iP-type/Z-type Cks are good indices of different developmentalstates in Prunus persica.     

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     

Cytokinin metabolism in Physcomitrella patens – differences and similarities to higher plants

Cytokinins play an important role in plant development and occur informs with different hormonal activity. As the nucleotide forms of cytokininsare considered to have little or no biological activity, the conversion ofcytokinin bases and ribosides to their nucleotides can contribute to the tuningof cytokinin activity in plant cells. Cytokinin metabolism was monitoredin vivo by feeding either radiolabelledisopentenyladenosine (³H-[9R]iP) or isopentenyladenine(³H-iP) to liquid grown chloronema tissue ofPhyscomitrellapatens (Hedw.) B.S.G. wild type. The riboside ³H-[9R]iPwas rapidly converted to ³H-iP, which was released into the culturemedium. The intracellular concentration of the ³H-iP was twice ashigh as extracellular. From the overall amount of ³H-iP about 95%were present in the medium. Cytokinin nucleotides occurred as tritiated mono-,di- and triphosphates of ³H-[9R]iP. When feeding the base³H-iP however, its main metabolic fate was degradation and nosignificant amounts of radiolabelled cytokinin nucleotides were detected. Forthe cytokinin metabolism in P. patens it is concludedthat,in contrast to higher plants nucleotides are mainly formed from ribosidesvia the adenosine kinase pathway and not byribophosphorylation of the cytokinin base via adeninephosphoribosyltransferase.     

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     

An evaluation of the role of cytokinins in the development of abnormal inflorescences in oil palms (Elaeis guineensis Jacq.) regenerated from tissue culture

Tissue cultures and regenerant plants from cell lines producing palms with normal and abnormal flowers were analyzed for cytokinin content and compared with zygotic embryos and seedlings. Immature inflorescences at the critical stage of flower development dissected from normal and abnormal palms were also analyzed. High performance liquid chromatography (HPLC)/radioimmunoassay and HPLC/enzyme-linked immunosorbent assay methods were used over a period of several years to measure the isoprenoid cytokinins. The results of analyses of endogenous aromatic cytokinins, present at comparable levels, will be reported separately. Oil palm cultures and regenerant plants contained relatively high concentrations of the 9-glucosides of isopentenyladenine ([9G]iP) and zeatin ([9G]Z). The predominant biologically active isoprenoid cytokinin present was zeatin riboside ([9R]Z), with lesser amounts of isopentenyladenine (iP) and isopentenyladenosine ([9R]iP). There was evidence of small amounts of dihydrozeatin compounds, but high concentrations (mainly as dihydrozeatin-9-glucoside ([9G]DHZ)) were confined to the haustorium of the zygotic embryo. Callus tissue contained very low concentrations of cytokinin. Frequently only [9G]iP could be detected, at about 1 pmol · g^-1 fresh weight, with [9R]Z at less than 0.05 pmol · g^-1. In comparison, nodular embryogenic tissues in vitro contained between 30 and 1,500 pmol · g^-1 of [9G]iP, 5–50 pmol · g^-1 of [9G]Z, and up to 12 pmol · g^-1 of [9R]Z. Shoots of regenerant plantlets and seedlings contained lower concentrations of [9G]iP (3–30 pmol · g^-1), although this was still the predominant cytokinin. [9R]Z and [9G]Z were present at between 2 and 15 pmol · g^-1, with iP at 1–5 pmol · g^-1 and [9R]iP at between 1 and 12 pmol · g^-1. Seedlings contained similar amounts with the exception of a lower [9G]iP content (5–10 pmol · g^-1) and more [9R]iP (10–20 pmol · g^-1). Root tissues of ramets contained significantly higher concentrations of [9G]iP than shoots. Comparison of two isogenic lines of one clone giving rise to normal and abnormal palms showed significantly higher concentrations of [9R]Z and [9G]Z in the normal than in the abnormal line and, in embryoids only, higher [9G]iP in the normal line. In all other cases the between-done differences were greater than any normal/abnormal differences. There was a general tendency for increased concentrations of [9G]iP in abnormal lines and for this compound to be in a higher concentration in embryoids and plants derived from culture than in zygotic embryos and seedlings. Analysis of cytokinins in immature female inflorescences of normal and abnormal palms of a single clone showed the abnormal inflorescences to have higher concentrations of [9R]Z and [9R]DHZ and less [9G]Z than the normal inflorescences at comparable stages of development.Abbreviations HPLC high performance liquid chromatography - [9G]iP 9-glucoside of isopentenyladenine - [9G]Z 9-glucoside of zeatin - [9R]Z zeatin riboside - iP isopentenyladenine - [9R]iP isopentenyladenosine - [9G]DHZ dihydrozeation-9-glucoside - ELISA enzyme-linked radioimmunosorbentassay - ANOVAR analysis of variance     

Hormones and Pod Development in Oilseed Rape (Brassica napus)

The endogenous levels of several plant growth substances (indole acetic acid, IAA; abscisic acid, ABA; zeatin, Z; zeatin riboside, [9R]Z; isopentenyladenine, iP; and isopentenyladenosine, [9R]iP were measured during pod development of field grown oilseed Rape (Brassica napus L. var oleifera cv Bienvenu) with high performance liquid chromatography and immunoenzymic (enzyme-linked immunosorbent assay, ELISA) techniques. Results show that pod development is characterized by high levels of Z and [9R]Z in 3 day old fruits and of IAA on the fourth day. During pod maturation, initially a significant increase of IAA and cytokinins was observed, followed by a progressive rise of ABA levels and a concomitant decline of IAA and cytokinin (except iP) levels. The relationship between hormone levels and development, especially pod number, seed number per pod, and seed weight determination, will be discussed.     

Thidiazuron influences the endogenous levels of cytokinins and IAA during the flowering of isolated shoots of Dendrobium

This study reports the effects of thidiazuron (TDZ) on the endogenous levels of indoleacetic acid (IAA), zeatin, zeatin riboside ([9R]Z), isopentenyladenine and isopentenyladenosine ([9R]iP) as well as structural changes in the apical meristem of Dendrobium Second Love shoots during flower induction and initial development in vitro. The results revealed that the presence of 1.8microM TDZ had a profound effect on the endogenous cytokinins (CKs) and IAA levels of the explants, when compared to those grown on a TDZ-free medium. A significant increase in CKs (especially [9R]iP and [9R]Z) and IAA in the first samples (taken at day 5) grown on TDZ-enriched medium was associated with flower induction, while a second increase in the level of these hormones after 25d of culture was related to flower development. The histological changes detected in the shoot apical meristem of explants grown in the presence of 1.8microM TDZ during 30d of culture are also described. Based on these findings, it is suggested that both auxin and CKs seem to be involved with the floral transition of Dendrobium Second Love in vitro. However, a possible direct effect of TDZ on flower formation is not discarded.     

Solid-phase extraction of cytokinins from aqueous solutions with C18 cartridges and their use in a rapid purification procedure

Eleven cytokinins-including bases, ribosides, glucosides, and ribotides-were tested for their retention on C₁₈ cartridges that were washed with 40 mL of water or a dilute acid at pH 3. Cytokinins were then eluted with methanol and analyzed by high performance liquid chromatography (HPLC). All pure cytokinin were well retained when the cartridge was washed with water, but Z and (diH)Z were less well retained at pH 3. The ribotides required 80% methanol for elution. Cotton leaf tissue (500 mg dry wt) was spiked with cytokinins, extracted with 80% methanol, and the extract bulk purified with hexane, insoluble polyvinylpyrrolidone, and minicolumns (strong anion exchange, amino, and C₁₈ cartridges). Ribotides, added to leaf tissue, could not be recovered as ribotides; it was necessary to hydrolyze and purify them as ribosides. The cytokinins were separated and analyzed by HPLC on strong cation exchange and C₁₈ columns. Recoveries through the entire procedure averaged 70%.Cytokinin abbreviations (diH)Z Dihydrozeatin - (diH)Z dihydrozeatin riboside - (diH)[9R]Z trans-zeatin - Z t-zeatin riboside - [9R]Z t-zeatin-O-glucoside - (OG)Z t-zeatin riboside-O-glucoside - (OG)[9R]Z t-zeatin riboside-5-monophosphate - [9R-5P]Z N6(^2-isopentenyl)adenine - iP N6(^2-isopentenyl)adenosine - [9R]iP N6(^2-isopentenyl)adenosine-5-monophosphate-[9R-5P]iP     

A rooting inhibitor present in Norway spruce seedlings grown at high irradiance - a putative cytokinin

Cuttings from seedlings of Picea abies L. (Karst.) grown under high light (HL). rooted slowly and incompletely, whereas rooting in cuttings from seedlings grown under low light (LL) was rapid and almost complete. Immunoaffinity chromatograpriy (IAC) with antibodies against isopentenyl adenosine ([9-RIiP) and trans-zeatin riboside (I9R]Z) was used to isolate cytokinin-like compounds from extracts of the cuttings. These compounds were separated by HPLC and quantified with a UV-detector. [9R]iP-type and [9R]Z-type compounds were also quantified using an enzyme immunoassay (ELISA). Levels of both [9R]iP-type and [9R]Z-type cytokinins were considerably higher in seedlings grown under HL than in those grown under LL. An unidentified compound (X), occurring in high amounts in the hypocotyls of HL seedlings and isolated by IAC and HPLC, inhibited rooting in cuttings from LL seedlings. Compound X was metabolised during the rooting period. Although X was retained on the imnimmoaffinity column, it did not bind to the antibodies to the degree necessary to allow quantification by ELISA. Treatment with acid phosphatase (EC 3.1.3.2) and β-glucosidase (EC 3.2.1.21) converted X into other unknown compounds. Some of these cross-ieacted with [9R)iP antibodies in ELISA.     

New synthesis of immunogenic N6-isopent-2-enyladenosine-protein conjugate. Preparation, purification, and specificity of related antibodies.

An original procedure which preserves the structure of the sugar ring is described to link a plant hormone as N6-isopent-2-enyladenosine [( 9R]iP) onto a protein carrier to prepare a more specific immunogen. This cytokinin is bound to bovine serum albumin (BSA) and ovalbumin by a five-step procedure. These [9R]iP-protein conjugates have a maximal absorption at 269 nm and show molar ratios of hormone bound to proteins in the range of 12:1 and 18:1 for BSA and ovalbumin, respectively. Polyclonal antibodies were raised in rabbits against [9R]iP-BSA and were purified by affinity chromatography. Titers and specificity of the antisera and purified antibodies were determined by ELISA and RIA. These antibodies are highly specific for [9R]iP and do not cross-react with zeatin and ribosylzeatin. An immunoaffinity matrix was prepared with a capacity of 1 microgram of [9R]iP/mL of gel.     

Benzyladenine and derivatives-their significance and interconversion in plants

Recently benzyladenine has been isolated as a natural cytokinin from a number of plants. The natural occurrence of this cytokinin will change the attitude with which physiologists view this hormone. This review attempts to put into context what is known about this cytokinin and its derivatives and to compare and contrast its metabolism and the function and physiological action of its various metabolites. Nothing is known about the biosynthesis of benzyladenine. Its structure would suggest that its biosynthetic pathway may differ considerably from that of zeatin and iso-pentenyladenine.Abbreviations Ade adenine - Ado adenosine - BA benzyladenine - [9R]BA BA ribonucleoside - [9R-MP]BA BA nucleotide - [9R-DP]BA BA dinucleotide - [9R-TP]BA BA trinucleotide - [3G]BA BA 3 glucoside - [7G]BA BA 7 glucoside - [9G]BA BA 9 glucoside - [9R-G]BA BA 9-ribosylglucoside - [9Ala]BA BA alanine-conjugate - (2OH)BA BA ortho-OH - (2OH)[9R]BA BA ortho-Oh-riboside - KN kinetin - [9R]KN KN ribonucleoside - DHZ dihydrozeatin - Z trans-zeatin - [9R]Z zeatin ribonucleoside - [7G]Z zeatin-7-glucoside - [9G]Z zeatin-9-glucoside - [9Ala]Z zeatin alanine-conjugate - (OG)[9R]Z O-glucoside of zeatin ribonucleoside - [9R-MP]Z zeatin nucleotide - iP iso-pentenyladenine - [9R]iP iP ribonucleoside     

Transport and metabolism of [3H]iso-pentenyladenine following application to the tips of intact and decapitated tap roots of Pinus pinea

[³H]iso-Pentenyladenine ([³H]iP) was fed for 24 h to the tips of intact and root tip-decapitated Pinus pinea seedlings. Twelve and 24 h after application to the roots of intact plants most of the applied radioactivity (±60%) was transported to the shoot. Root tip removal increased transport of the applied radioactivity to the shoot, but the overall pattern of distribution of radioactivity in the seedling did not change. Large amounts of radioactivity were recovered from the elongation zone of the root. Some radioactivity also accumulated in the older part of the root with well-developed lateral roots. When [³H]iP was applied one day after decapitation, no significant changes in the pattern of radioactivity distribution were found between the intact and decapitated root systems. However, when applied 7 days after decapitation there was a significant increase of radioactivity in the region of the root where lateral roots were emerging. HPLC separation of extracts from the different root sections showed that [³H]iP was extensively metabolized in the root. Six peaks of radioactivity, which co-chromatographed with authentic cytokinin standards, were detected.Abbreviations ABA abscisic acid - ADE adenine - IAA indole-acetic acid - iP iso-pentenyladenine - HPLC high performance liquid chromatography - [OG]DHZ O-glycosyldihydrozeatin - [9R-MP]DHZ ribosyldihydrozeatin monophosphate - [9G]iP iso-pentenyladenine-9-glucoside - [9R]Z ribosylzeatin - [9R]iP iso-pentenyladenosine - TLC thin layer chromatography     

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.     

Quantification by ELISA of Cytokinins in Root-Pressure Exudates of Urtica dioica Plants Grown under Different Nitrogen Levels

ELISAs for [9R]iP, [9R]Z and (diH) [9R]Z have been worked out which, in combination with HPLC., allow the quantitative determination of cytokinins of the isopentenyladenin-, zeatin- and dihydrozeatin-type in samples of plant material. The three ELISAs are optimally applicable in the range from 0.5 to 20 pmoles of cytokinins per assay but reliable measurements can still be performed down to 0.05 pmoles. The assay has been applied to root pressure exudates obtained from Urtica dioica plants grown under controlled conditions and at different nitrogen (nitrate) levels. The predominant cytokinins of the xylem exudate were Z, Z-nucleotide, iP, [9R]iP and iP-nucleotide, while (diH)Z was present to a minor extent. The concentrations of these compounds were found to range between 0.2 and 3 pmoles per ml exudate. Interestingly, no effect of the different nitrogen nutrition on the pattern and concentration of cytokinins in the xylem exudate could be established.     

Cultivars of Hexaploid Wheat of Contrasting Stature and Chlorophyll Retention Differ in Cytokinin Content and Responsiveness

The work reported here compared cytokinin content and sensitivityin a selection of hexaploid wheat (Triticum aestivum L.) cultivarsusing the following measurements: leaf cytokinins at three timepoints during light-growth and at four 24 h intervals afterlight-grown plants were transferred to darkness; sensitivityof root growth to direct applications of isopentenyl adenosine([9R]iP); and, sensitivity of germination and subsequent rootand shoot growth to 18 h imbibition of seeds in benzyladenine(BA). Accumulation of zeatin riboside-type cultivars was greatestduring light-growth in Tibet Dwarf, a wheat with an extremedwarf phenotype, intermediate in Omar standard and dwarf cultivars,and lowest in the standard and dwarf versions of Itana. Cytokininlevels were otherwise not directly correlated to plant staturein these wheats. There were no cultivar-associated qualitativedifferences in the types of cytokinins detected in this study.During the 16 h light period, the content of zeatin riboside-typecytokinins increased up to tenfold and then declined to basallevels during dark growth. Chlorophyll retention during dark-growthwas correlated with leaf cytokinin content. Data collected ata restricted number of sampling points during dark-growth suggesteda cyclic accumulation of [9R]iP-type cytokinins and the apparentcycle in Tibet Dwarf was offset by 24 h. Tibet Dwarf showedthe greatest root growth inhibition after exposure of seedlingroots to [9R]iP or imbibition of seeds in BA. Neither of thesetreatments affected shoot growth in any of the cultivars. Wheat; Triticum aestivum ; cytokinin; zeatin riboside; benzyladenine; root inhibition     

Thermoperiodic effect on flowering and endogenous hormonal status in Dendrobium (Orchidaceae)

Two year-old cloned plants of Dendrobium Second Love were submitted to 25 °C (light) and 10 °C (dark) under a 12 h photoperiod (60 μmol m⁻¹ s⁻¹) for 30 days. The endogenous levels of IAA, ABA, and the cytokinins Z, [9R]Z, iP, and [9R]iP were measured 15, 22, and 30 days after the start of the thermoperiodic treatment in lateral buds and leaves. The endogenous levels of IAA and cytokinins, especially the zeatin-derived forms, increased significantly in buds after 15 days of treatment. On the other hand, the amount of ABA decreased progressively and significantly throughout the treatment. The treatment conspicuously accelerated flower-bud development. The found correlation suggests that hormones are involved in the signal transduction pathway of thermoperiodic flowering control.     

Arabidopsis SOI33／AtENT8 Gene Encodes a Putative Equilibrative Nucleoside Transporter That Is Involved in Cytokinin Transport In Planta

The plant phytohormone cytokinin plays an important role in many facets of plant growth and development by regulating cell division and differentiation. Recent studies have shed significant light into the mechanisms of cytokinin metabolism and signaling. However, little is known about how the hormone is transported in planta, although it has been proposed that the hormone is presumably transported in nucleoside-conjugated forms. Here, we report the identification and characterization of cytokinin transporters in Arabidopsis. We previously reported that a gain-of-function mutation in the PGA22/AtlPT8 gene caused overproduction of cytokinins in planta. In an effort to screen for suppressor of pga22/atipt8 (soi) mutants, we identified a mutant soi33-1. Molecular and genetic analyses indicated that S0133 encodes a putative equilibrative nucleoside transporter (ENT), previously designated as AtENT8. Members of this small gene family are presumed to be involved in the transport of nucleosides in eukaryodc cells. Under conditions of nitrogen starvation, loss-of-function mutations in SOI33/AtENT8 or in a related gene AtENT3 cause a reduced sensitivity to the nucleoside-type cytokinins isopentenyladenine riboside (iPR) and transzeatin riboside (tZR), but display a normal response to the free base-type cytokinins isopentenyladenine (iP) and trans-zeatin (tZ). Conversely, overexpression of SOI33/AtENT8 renders transgenic plants hypersensitive to iPR but not to iP. An in planta measurement experiment indicated that uptake efficiency of^3Hlabeled iPR was reduced more than 40% in soi33 and atent3 mutants. However, a mutation in AtENT1 had no substantial effect on the cytokinin response and iPR uptake efficiency. Our results suggest that SOI33/AtENT8 and AtENT3 are involved in the transport of nucleoside-type cytokinins in Arabidopsis.     

Arabidopsis SOI33/AtENT8 Gene Encodes a Putative Equilibrative Nucleoside Transporter That Is Involved in Cytokinin Transport In Planta

The plant phytohormone cytokinin plays an important role in many facets of plant growth and development by regulating cell division and differentiation. Recent studies have shed significant light into the mechanisms of cytokinin metabolism and signaling. However, little is known about how the hormone is transported in planta, although it has been proposed that the hormone is presumably transported in nucleoside-conjugated forms. Here, we report the identification and characterization of cytokinin transport ers in Arabidopsis. We previously reported that a gain-of-function mutation in the PGA22/AtIPT8 gene caused overproduction of cytokinins in planta. In an effort to screen for suppressor of pga22/atipt8 (soi) mutants, we identified a mutant soi33-1. Molecular and genetic analyses indicated that SOI33 encodes a putative equilibrative nucleoside transporter (ENT), previously designated as AtENT8. Members of this small gene family are presumed to be involved in the transport of nucleosides in eukaryotic cells. Under conditions of nitrogen starvation, loss-of-function mutations in SOI33/AtENT8 or in a related gene AtENT3 cause a reduced sensitivity to the nucleoside-type cytokinins isopentenyladenine riboside (iPR) and trans zeatin riboside (tZR), but display a normal response to the free base-type cytokinins isopentenyladenine (iP) and trans-zeatin (tZ). Conversely, overexpression of SOI33/AtENT8 renders transgenic plants hyper sensitive to iPR but not to iP. An in planta measurement experiment indicated that uptake efficiency of 3H labeled iPR was reduced more than 40% in soi33 and atent3 mutants. However, a mutation inAtENT1 had no substantial effect on the cytokinin response and iPR uptake efficiency. Our results suggest that SOI33/ AtENT8 and AtENT3 are involved in the transport of nucleoside-type cytokinins in Arabidopsis.     

In situ localisation of cytokinins in the shoot apical meristem of <Emphasis Type="Italic">Sinapis alba</Emphasis> at floral transition

In plants of Sinapis alba L. induced to flower by one long day (LD), previous work showed that the phloem sap feeding the shoot apex is enriched in cytokinins of the isopentenyladenine (iP)-type between 9 and 25 h after start of the LD [P. Lejeune et al. (1994) Physiol Plant 90:522-528]. We have checked the hypothesis that the cytokinin content of the shoot apical meristem (SAM) should increase in response to floral induction by one LD using histoimmunolocalisation techniques and rabbit antiserum against isopentenyladenosine or zeatin riboside. The free bases iP and zeatin are present only in apical tissues containing dividing cells. At 30 h after the start of an inductive LD, a markedly increased iP immune reaction is observed in SAM tissues while the level of zeatin is not modified. Our results are in line with the data obtained by analysis of phloem sap.     

Cytokinin oxidase from auxin- and cytokinin-dependent callus cultures of tobacco (Nicotiana tabacum L.)

Cytokinin oxidase was extracted and partially purified from auxin- and cytokinin-dependent callus tissue of tobacco (Nicotiana tabacum L. cv. Wisconsin 38). The activity of the enzyme preparation was examined using an assay based on the conversion of tritiated N⁶-(²-isopentenyl)adenine ([2,8-³H]iP) to adenine. Cytokinin oxidase exhibited a temperature optimum at 45–50°C and a relatively high pH optimum (8.5–9.0). The apparent K_m value of the enzyme was 4.3 M for iP. On the basis of the substrate competition assays, iP was determined to be the preferred substrate of the enzyme. Substrate competition was also observed with zeatin and the cytokinin-active urea derivative Thidiazuron. Cytokinins bearing saturated isoprenoid side chains or cyclic side chain structures, as well as auxins and abscisic acid, had no effect on the conversion of [2,8-³H]iP. The cytokinin oxidase exhibited increased activity in the presence of copper-imidazole complex in the reaction mixture. Under optimal concentrations of copper (15 mM CuCl₂) and imidazole (100 mM), the enzyme activity was enhanced ca. 40-fold. Under these conditions the pH optimum was lowered to pH 6.0, whereas the temperature optimum, the apparent K_m value, and the substrate specificity were not altered. Most of the enzyme moiety did not bind to the lectin concanavalin A. The characteristics of cytokinin oxidase presented here suggest that a novel molecular form of the enzyme, previously identified and characterized in Phaseolus lunatus callus cultures (Kamínek and Armstrong (1990) Plant Physiol 93:1530), also occurs in cultured tobacco tissue.Abbreviations Ade adenine - iP N⁶-(²-isopentenyl)adenine - [2,8-³H]iP [2,8-³H]-N⁶-(²-isopentenyl)adenine - [9R]iP N⁶-(²-isopentenyl)adenosine - (diH)iP N⁶-isopentyladenine - (diH)Z dihydrozeatin - BAP N⁶-benzyladenine - ( _o OH)[9R]BAP N⁶-(o-hydroxybenzyl)adenosine - (mOH)[9R]BAP N⁶-(m-hydroxybenzyl)adenosine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA naphthalene-1-acetic acid - ABA abscisic acid - Con A concanavalin A