Accumulation of zeatin O-glycosyltransferase in Phaseolus vulgaris and Zea mays following cold stress

Zeatin O-glycosides have been reported as inactive and stable storage forms of cytokinins whose concentrations increase in cold stressed plants. Zeatin O-glycosides accumulation in developing bean seeds has been correlated with an increase of zeatin O-glycosyltransferase , which is specific to trans-zeatin, and catalyzes the conjugation of zeatin O-glycosides. When Phaseolus vulgaris and Zea mays seedlings were grown for 3 days at 25 and then incubated at 4 or 10 for 6 days no further growth was observed in roots. Hypertrophy was observed in the root tips of both species. In shoot-hypocotyl complexes, in contrast, growth occurred when seedlings were incubated at 10 . Western analysis, with Mabs specific to zeatin O-glycosyltransferase, detected antigenically related proteins in roots, shoot tips and cotyledons after seedlings were cold stressed for 1–6 days at 4 or 10 . Immunolocalization, of both maize and bean root sections grown at 25 revealed antigenically related proteins that were detected at low levels in cortical cells. The signal intensified upon cold stress. The localization of zeatin O-glycosyltransferase in Z. mays root tips was directly comparable to the distribution of the zeatin O-glycosides. The enzyme was detected in the nucleus, cytoplasm, and closely associated with the plasma membrane and in the cell wall of Z. mays root cells. Southern analysis suggested that more than one gene in Z. mays that were homologous to zeatin O-glycosyltransferase in P. vulgaris. Zeatin O-glycosyltransferase may be involved in modulation of cytokinins under cold stress.     

Crystal structure of Vigna radiata cytokinin-specific binding protein in complex with zeatin

The cytosolic fraction of Vigna radiata contains a 17-kD protein that binds plant hormones from the cytokinin group, such as zeatin. Using recombinant protein and isothermal titration calorimetry as well as fluorescence measurements coupled with ligand displacement, we have reexamined the K(d) values and show them to range from approximately 10(-6) M (for 4PU30) to 10(-4) M (for zeatin) for 1:1 stoichiometry complexes. In addition, we have crystallized this cytokinin-specific binding protein (Vr CSBP) in complex with zeatin and refined the structure to 1.2 A resolution. Structurally, Vr CSBP is similar to plant pathogenesis-related class 10 (PR-10) proteins, despite low sequence identity (<20%). This unusual fold conservation reinforces the notion that classic PR-10 proteins have evolved to bind small-molecule ligands. The fold consists of an antiparallel beta-sheet wrapped around a C-terminal alpha-helix, with two short alpha-helices closing a cavity formed within the protein core. In each of the four independent CSBP molecules, there is a zeatin ligand located deep in the cavity with conserved conformation and protein-ligand interactions. In three cases, an additional zeatin molecule is found in variable orientation but with excellent definition in electron density, which plugs the entrance to the binding pocket, sealing the inner molecule from contact with bulk solvent.     

A gene encoding the cytokinin enzyme zeatin O-xylosyltransferase of Phaseolus vulgaris.

Zeatin is the most active and ubiquitous form of the naturally occurring cytokinins. Glycosyl conjugates of zeatin are found in many plant tissues and are considered important for storage and protection against degradative enzymes. Two enzymes catalyzing the formation of O-glycosyl derivatives of zeatin have been characterized, O-glucosyltransferase and O-xylosyltransferase, occurring in seeds of lima bean (Phaseolus lunatus) and bean (Phaseolus vulgaris), respectively. Recently, the ZOG1 gene (zeatin O-glucosyltansferase) was isolated from P. lunatis (). Based on the ZOG1 sequence, the ZOX1 gene (zeatin O-xylosyltransferase) was cloned from P. vulgaris. ZOX1 contains an open reading frame of 1362 bp that codes for a 454-amino acid peptide of 51 kD. The recombinant protein has properties identical to the native enzyme: it catalyzes O-xylosylzeatin formation with UDP-Xyl as a glycosyl donor but does not recognize UDP-Glucose as a substrate. The ZOX1 and ZOG1 genes exhibit 93% identity at the nucleotide level and 90% similarity at the amino acid level. Neither gene contains introns. These zeatin-specific genes and their promoters will be useful for studies of the regulation of active versus storage forms of cytokinins. Comparison of sequences encoding similar enzymes with distinct substrate specificity may lead to identification of epitopes specific to cytokinin and glycosyl donor molecules.     

Substrate specificity and domain analyses of zeatin O-glycosyltransferases

The substrate specificity of two recombinant enzymes, zeatin O-glucosyltransferase 1 (ZOG1) and zeatin O-xylosyltransferase 1 (ZOX1), was further characterised. ZOG1 utilises zeatin (Z), UDPG, and UDPX as substrates to form O-glucosylzeatin (OGZ) and O-xylosylzeatin (OXZ) but has higher affinity to UDPG than UDPX. ZOX1 uses only UDPX, converting Z to OXZ. Dihydrozeatin (DHZ) is also a substrate for both enzymes, but only in combination with UDPX, giving rise to O-xylosyldihydrozeatin (OXDHZ). O-Glucosyldihydrozeatin (OGDHZ) is not formed by ZOG1, possibly due to steric hindrance. Regions relevant to UDPG/UDPX affinity and competition were identified using hybrid enzymes derived from domain exchanges of parental genes. The N-terminal half of the enzyme is important in this respect. The BstEII-BstAPI segment of ZOG1 correlates with inhibition of O-xylosyltransferase activity by UDPG while the BstAPI-Eco0109 segment of ZOG1 is required for utilisation of UDPG as the sugar donor.     

Structure of mistletoe lectin I from Viscum album in complex with the phytohormone zeatin

The crystal structure of mistletoe lectin I (ML-I) isolated from the European mistletoe Viscum album in complex with the most active phytohormone zeatin has been analyzed and refined to 2.54 A resolution. X-ray suitable crystals of ML-I were obtained by the counter-diffusion method using the Gel-Tube R crystallization kit (GT-R) onboard the Russian Service Module on the international space station ISS. High quality hexagonal bipyramidal crystals were grown during 3 months under microgravity conditions. Selected crystals were soaked in a saturated solution of zeatin and subsequently diffraction data were collected applying synchrotron radiation. A distinct F(o)-F(c) electron density has been found inside a binding pocket located in subunit B of ML-I and has been interpreted as a single zeatin molecule. The structure was refined to investigate the zeatin-ML-I interactions in detail. The results demonstrate the ability of mistletoe to protect itself from the host transpiration regulation by absorbing the most active host plant hormones as part of a defense mechanism.     

An enzyme mediating the conversion of zeatin to dihydrozeatin in phaseolus embryos

A reductase catalyzing the conversion of zeatin to dihydrozeatin was detected in soluble fractions of immature Phaseolus vulgaris embryos. The enzyme was partially purified by ammonium sulfate fractionation and affinity, gel filtration, and anion exchange chromatography. NADPH was the only cofactor required for enzyme activity, and the pH optimum was 7.5 to 8.0. The enzyme did not recognize compounds closely related to zeatin, such as ribosylzeatin, cls-zeatin, O-xylosylzeatin, N⁶-(Δ²-isopentenyl)adenine, or N⁶-(Δ²-isopentenyl)adenosine. No conversion of dihydrozeatin to zeatin by the enzyme was observed. Two forms of the reductase could be separated by either gel filtration or anion exchange high performance liquid chromatography. The high molecular weight isozyme (M_r 55,000 ± 5,000) eluted as the second peak from the anion exchange column, while the low molecular weight isozyme (M_r 25,000± 5000) was less negatively charged. The results suggest that side chain reduction occurs at the free base level. In addition, Phaseolus embryos are useful for the detection of zeatin-specific metabolic enzymes.     

The effect of sucrose on the levels of abscisic acid, indoleacetic acid and zeatin/zeatin riboside in wheat ears growing in liquid culture

The influence of a varied sucrose supply on grain size and hormonal contents of detached wheat ears ( Triticwn aestivum L. cv. Schirokko) was investigated throughout grain development. In ears led limited amounts, or no sucrose, grain weights in both proximal and distal grain positions of the ear were reduced. Radioimmunoassay for abscisic acid, indoleacetic acid and zeatin/zeatin riboside showed that the changes in the levels of these hormones in grains and bracts were comparable to intact ears when detached ears were well supplied with sucrose. Under conditions of limited sucrose supply, higher abscisic acid levels in the distal and proximal grains of detached ears were found compared to ears supplied with adequate sucrose. Limiting sucrose supply to the ear did not alter the levels of indoleacetic acid or zeatin/zeatin riboside in either the grains or bracts of detached ears.     

A New Method for Differential Quantitative Assay of Zeatin and Zeatin Riboside

A new method is proposed for differential quantitative assay of two major endogenous cytokinin forms. It is based on determination of two effective parameters—concentrations of zeatin and zeatin riboside—with the use of appropriate antigens as standards. The method can be used for determining cytokinins in small samples of plant tissues without extract fractionation. This study pioneers the quantitation of changes in the hormonal status of ovules and ovaries of Triticum aestivumL. at early stages of embryogeny. A gradual increase in the content of the active and storage forms of the hormones from the ovary to the ovule was revealed.     

A kinetic analysis of the effects of gibberellic Acid, zeatin, and abscisic Acid on leaf tissue senescence in rumex

Hormones which inhibit senescence in Rumex leaf tissue in the dark include gibberellic acid and the cytokinin zeatin. Abscisic acid accelerates senescence in this tissue. Other workers have proposed that cytokinins, but not gibberellins, interact with abscisic acid in senescing Rumex leaf tissue. The present study reinvestigates the question of interaction using measurements of chlorophyll degradation kinetics as parameters of senescence rate and draws the conclusion that neither zeatin nor gibberellic acid interact with abscisic acid in this system. In support of this conclusion are these results. Zeatin clearly cannot overcome the effects of abscisic acid when hormone solutions are replaced every other day. The kinetics of chlorophyll breakdown for tissue treated with unreplaced saturating zeatin solutions is different from that of tissue exposed to saturating zeatin plus abscisic acid. The observed rates of chlorophyll breakdown for tissue treated with abscisic acid and zeatin agree closely with predicted rates using a multiplicative model for independent action of the two hormones.     

Mass spectrometric measurement of zeatin glycoside levels in Vinca rosea L. crown gall tissue

The range of zeatin glycosides found in crown gall tissue of Vinca rosea L. has been quantified using a mass spectrometric isotope dilution procedure. Problems in the quantitative analysis of cytokinins in plant extracts are discussed.Abbreviations GC/MS coupled gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - Me methyl - Z zeatin - Z9G zeatin 9-glucoside - ZOG zeatin O-glucoside - ZR zeatin 9-riboside - ZROG zeatin 9-riboside O-glucoside     

Quantitation of cytokinins in biological samples using antibodies against zeatin riboside

The cross-reactivity of antibodies elicited in rabbits against zeatin riboside, to a wide range of naturally occurring cytokinins, was examined. As well as to zeatin riboside, the antisera cross-reacted to a considerable extent with zeatin, lupinic acid, zeatin-9-glucoside, zeatin riboside 5′-monophosphate and to a much lesser, but measurable extent, with dihydrozeatin riboside and dihydrozeatin. Chromatographic methods were devised which allowed separation of all these cross-reactive compounds. Four biological samples, extracts of immature Zea mays kernels, immature seeds of Lupinus luteus, and Datura innoxia crown gall tumor tissue, and a sample of Agrobacterium tumefaciens culture supernatant, were purified by these chromatographic methods, using [³H]zeatin riboside as a recovery marker, and at each stage of the purification process, were subjected to radioimmunoassay over a range of dilutions. At each stage of sample purification, sample dilution curves were found to be parallel to the standard curve. Sample cytokinin levels estimated by radioimmunoassay were in close agreement to those available in the literature for similar samples assayed by alternative methods. However, in some samples, unknown cross-reacting compounds were detected.     

Comparison of zeatin indoleacetate with zeatin and indoleacetic Acid in the tobacco bioassay

Zeatin indole-3-acetate, 6-[4-(indole-3-acetoxy)-3-methyl-trans-2-butenylamino]purine, is at least as effective as zeatin on a molar basis in satisfying the cytokinin requirement for growth and bud formation in tobacco bioassays. It is less effective than indole-3-acetic acid and is needed as a variable function of the cytokinin concentration for satisfying the optimal requirement of an auxin. Comparisons of the types of growth and yield of tissue obtained with serial concentration of the ester and with equimolar mixtures of its free base and acid indicate that the relative requirement for auxin changes with the concentration of cytokinin and is related to the types of callus growth and differentiation which occur. The results also suggest that the ester serves as a source of auxin only after modification, presumably by hydrolysis to indoleacetic acid.     

Development of transgenic tobacco harboring a zeatin O-glucosyltransferase gene from Phaseolus

Summary Zeatin and its derivatives are major consituents of higher plant cytokinins. Metabolic steps modifying the isoprenoid side chain, such as O-glycosylation, are expected to have a direct bearing on cytokinin-mediated processes. To examine this possibility, transgenic tobacco plants were generated harboring a gene (ZOG1) encoding a zeatin O-glucosyltransferase from Phaseolus lunatus under the control of a constitutive (35S) and an inducible (Tet) promoter. The presence of the transgene resulted in elevated enzyme production and conversion of exogenous zeatin to its O-glucoside, confirming the expression of the ZOG1 gene in transgenic plants. Endogenous O-glucosylzeatin was increased from less than 1 pmol per g fresh weight in leaves and roots of controls to 26 and 68 pmol per g fresh weight in leaves and roots of 35S-ZOG1 transformants, respectively. In cytokinin/auxin interaction experiments, Tet-ZOG1 leaf discs, in the presence of tetracycline, required 10-fold higher zeatin concentrations for the formation of shoots and callus than the controls. In 35S-ZOG1 plants, developmental changes included adventitious root formation on the lower stems, shorter stature, and axillary shoot growth. Thus, increased zeatin O-glucosylation in detached, cytokinin-dependent tissues leads to a shift in the response to exogenous zeatin indicative of cytokinin sequestering. In whole plants the effect can simulate a reduction or a rise in cytokinin activity depending on the tissue and stage of development. The use of tissue- and stagespecific promoters in the future will allow more precise analyses and targeted growth alterations.     

Identification of abscisic acid glucose ester,indole-3-acetic acid,zeatin and zeatin riboside in receptacles of pear

The identity of abscisic acid glucose ester, indole acetic acid, zeatin, and its riboside in pear receptacles was revealed by use of chromatographic, ultraviolet and mass spectral analysis.     

Identification of zeatin and zeatin riboside in cones of the hop plant and their possible role in cone growth

Cytokinin contents of fertilized cones, unfertilized cones andseeds of the hop plant (Humulus lupulus L.) were found to be0.08 ppm (Benzyladenine equivalent), 0.03 ppm and 0.5 ppm respectively.Major cytokinin in the former two was determined to be zeatinriboside based on gas chromatography-mass spectrometry afterseveral purification steps. Two more cytokinins were detectedin fertilized cones, one of which was determined to be zeatin.The possible role of endogenous cytokinin in cone growth isdiscussed in terms of quantitative and qualitative data obtained.Furthermore, effective purification techniques introduced inthis work are also discussed. (Received December 2, 1977; )     

Changes in the hormonal status of the Taraxacum officinale Web. ovary at early stages of embryogenesis

The hormonal status of the Taraxacum officinale Web. ovary was quantitatively assayed for the first time during early stages of embryogenesis. Apparent concentrations of endogenous cytokinins were measured using two systems of enzyme-linked immunosorbent assay (ELISA). The ELISA systems differed from one another by the specificity for the main endogenous forms of zeatin. The specificity of two heterological ELISA systems based on zeatin- and kinetin-specific antisera was studied. A new immunochemical approach to the problem of differential quantitative determination of natural zeatin forms is suggested. This approach does not require preliminary separation of experimental samples into individual fractions. True concentrations of zeatin and zeatin riboside in the T. officinale ovary were calculated based on the average values of apparent concentrations of endogenous cytokinins. When the embryo sac maturation had been completed, there was a threefold increase in the zeatin riboside concentration within the following 12 h. By the time of the first division of an unfertilized ovicell (i.e., within the next 12 h), there had been a twofold decrease in the zeatin riboside concentration. Therefore, at early stages of division of the unfertilized ovicell the zeatin riboside concentration virtually returned to the initial level. In contrast to zeatin riboside, there was a steady trend toward an increase in the zeatin concentration in the T. officinale ovary. Within the first 12 h and the next 12 h after completion of the embryo sac maturation, the zeatin concentration was increased 1.5-fold and 2-fold, respectively. The results of this work provide a pioneering insight into the dynamics of various natural forms of zeatin during the reproductive process. The immunochemical approach to quantitative monitoring of various natural forms of zeatin and their dynamics during embryogenesis suggested in this work can be extended to similar biological, medical, and agricultural problems of differential determination of low-molecular-weight agents of similar structure but different biological activity.     

Cytokinin translocation: Changes in zeatin and zeatin-riboside levels in the root exudate of tomato plants during their development

Summary The zeatin and zeatin riboside content of tomato (Lycopersicon esculentum Mill.) root exudates were determined at different stages of development. Zeatin riboside was found to be the major translocational form of cytokinin in the xylem during early vegetative growth. During flower bud formation this cytokinin decreased markedly in concentration so that, at anthesis, there was no appreciable difference in the zeatin and zeatin riboside concentration in the root exudate.     

Cytokinin metabolism in phaseolus embryos : genetic difference and the occurrence of novel zeatin metabolites

The metabolism of trans-[8-¹⁴C]zeatin was examined in embryos of Phaseolus vulgaris cv Great Northern (GN) and P. lunatus cv Kingston (K) in an attempt to detect genetic variations in organized plant tissues. Metabolites were fractionated by HPLC, and identified by chemical and enzymic tests and GC-MS analyses. Five major metabolites were recovered from P. vulgaris embryo extracts: ribosylzeatin, ribosylzeatin 5′-monophosphate, an O-glucoside of ribosylzeatin, and two novel metabolites, designated as I and II. Based on results of degradation tests and GC-MS analyses, I and II were tentatively identified as O-ribosyl derivatives of zeatin and ribosylzeatin. In embryos of P. lunatus, however, metabolites I and II were not present. The major metabolites were ribosylzeatin, ribosylzeatin 5′-monophosphate, and the O-glucosyl derivatives of zeatin and ribosylzeatin. The zeatin metabolites recovered were the same for embryos of different sizes but their quantities varied with embryo size and incubation time. The genetic differences appear to be embryo-specific and may be useful in the studies of the possible relationship between abnormal interspecific hybrid embryo growth and hormonal derangement in Phaseolus. In addition, analyses of both organized (intact) and unorganized (callus) tissues of the same genotype may provide an opportunity to address the problem of differential expression of genes regulating cytokinin metabolism during plant development.     

Interference from xylem sap in an enzyme-linked immunosorbent assay for zeatin riboside

The extent of interference from xylem sap in an enzyme-linked immunosorbent assay was determined for a woody perennial [ Populus trichocarpa Torr. & Gray x P deltoides Bart, ex Marsh (Hybrid 1l–ll)] and a herbaceous annual ( Phasesolus vulgaris L. cv. Contender). Crude xylem sap collected from excised roots from both species interfered with the assay for zeatin riboside. Assays for zeatin riboside in xylem sap collected from Popidus overestimated endogenous levels, and added standards could not be accurately measured from a range of sap dilutions. When Phaseolus plants were grown under various nutrient regimens, interference in the assay was dependent on nutrient availability. Of xylem sap components (inorganic minerals, amino acids and sucrose) which may vary with environmental conditions or among species, only sucrose interfered at the concentrations tested. Since the pH of xylem sap varies it was necessary to buffer samples prior to analysis. Partial purification using anion exchange columns and Sep-Paks cffectively eliminated interference. These results demonstrate that estimates of plant growth regulators in xylem sap by the ELISA (enzyme-linked immunosorbant assay) method can be influenced by species and environmental conditions such as plant nutritional status.     

Identification and quantitation by GC-MS of zeatin and zeatin riboside in xylem sap from rootstock and scion of grafted apple trees

Zeatin and zeatin riboside were identified by full-scan gas chromatography-mass spectrometry (GC-MS) in xylem sap of clonal apple rootstocks (M.27, M.9 and MM.106). These rootstocks exhibit a wide range of control over tree size when grafted to a common scion. The concentrations of zeatin and zeatin riboside were measured by GC-MS selected ion monitoring (SIM) in shoot xylem sap and root pressure exudate obtained from these rootstocks and from trees of Fiesta scion grafted onto the rootstocks. Zeatin was the predominant cytokinin in xylem sap from the dwarfing rootstocks, M.27 and M.9, while zeatin riboside was the predominant cytokinin in xylem sap from the more invigorating rootstock MM.106. Cytokinin concentrations (ng ml^–1) in root pressure exudate and shoot xylem sap, (i.e. from above the graft union in composite trees), increased with increasing vigour of the rootstock, irrespective of whether the plants were non-grafted rootstocks, or were composite plants of Fiesta scion grafted onto the rootstocks. Cytokinin content (ng shoot^–1) of shoot sap differed with rootstock; the more invigorating (MM.106) had greater amounts of cytokinins than the more dwarfing (M.9 and M.27) rootstocks. These results are discussed in relation to possible influences of roots on the growth of shoots via cytokinin supplies in the xylem sap.