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.     

Metabolism of C-zeatin in phaseolus embryos : occurrence of o-xylosyldihydrozeatin and its ribonucleoside

The metabolism of trans-[8-¹⁴C]zeatin was examined in embryos of Phaseolus acutifolius A. Gray P.I. 321637 and Phaseolus coccineus Lam. cvs Scarlet Runner and Desiree. In both species zeatin was converted to ribosylzeatin, ribosylzeatin 5′-monophosphate, O-glucosyl-9-ribosylzeatin and the recently discovered O-xylosyl derivatives of zeatin and ribosylzeatin (Turner, JE, DWS Mok, MC Mok, G Shaw 1987 Proc Natl Acad Sci USA. In press). Two new metabolites, identified by enzyme degradation and gas chromatography-mass spectrography analyses as O-xylosyldihydrozeatin and its ribonucleoside, were recovered from P. coccineus embryos. From this and previous studies it may be concluded that the potential to form O-xylosyl derivatives of zeatin is present only in embryos of three Phaseolus species (P. vulgaris L., P. coccineus, and P. acutifolius), but not in P. lunatus L., while the reduction of the side chain is most prominent in P. coccineus.     

The complex of O-glucosylzeatin derivatives formed in Populus species

When zeatin was supplied to excised leaves of Populus alba, the principal metabolites formed were adenosine, O-β-d-glucopyranosyl-cis-zeatin (derived from cis-zeatin in the commercial zeatin used), O-β-d-glucopyranosylzeatin, and two new metabolites, namely, O-β-d-glucopyranosyldihydrozeatin and O-β-d-glycopyranosyl-9-β-d-ribofuranosyldihydrozeatin, the structures of which were confirmed by unambiguous synthesis. Chromatographic studies indicated that adenosine 5′-phosphate, zeatin 7-glucopyranoside, zeatin 9-glucopyranoside, dihydrozeatin and zeatin 9-riboside were minor metabolites. The principal metabolites of zeatin 9-riboside in P. nigra leaves were the new metabolites O-β-d-glucopyranosyl-9-β-d-ribofuranosylzeatin (synthesized chemically) and O-β-d-glucopyranosl-9-β-d-ribofuranosyldihydrozeatin.     

A monoclonal antibody specific to zeatin o-glycosyltransferases of phaseolus

Zeatin O-xylosyltransferase and zeatin O-glucosyltransferase occur in immature embryos of Phaseolus vulgaris and P. lunatus, respectively. Purified preparations of the xylosyltransferase were used as antigen to elicit the formation of antibodies in mice. Hybridoma clones were produced by fusion of mouse spleen cells with myeloma cell line Fox-NY. A clone secreting monoclonal antibody (MAb), XZT-1, capable of immunoprecipitating both enzymes was obtained. The MAb detected a unique protein band from crude embryo extracts of each species with the correct molecular mass (50 kilodaltons) and relative charge (R_F = 0.5 and 0.3) of the respective enzymes. Competition experiments with substrates indicated that the glycosyl dinucleotide binding sites of the enzymes are probably not involved in MAb-enzyme recognition. Western blotting of samples from vegetative tissues of P. vulgaris detected a low level of O-glucosyltransferase but not O-xylosyltransferase, in leaves. These findings suggest the occurrence of two genes in P. vulgaris coding for O-glycosylation enzymes with tissue-specific expression. The MAb will be used to screen expression libraries and to obtain pure enzymes for amino acid sequencing and for the production of additional MAbs.     

Identification of Cytokinins from Xylem Exudate of Phaseolus vulgaris L

The principal biologically active cytokinins in xylem exudate of young Phaseolus vulgaris L. plants were identified by bioassay, high-performance liquid chromatography, enzymic degradation and combined gas chromatography-mass spectrometry (selected ion monitoring) a zeatin riboside, zeatin nucleotide, dihydrozeatin riboside, dihydrozeatin nucleotide, O-glucosyl zeatin, O-glycosyl dihydrozeatin, O-glucosyl dihydrozeatin riboside, and O-glucosyl dihydrozeatin nucleotide. Trace amounts of O-glucosyl zeatin riboside and O-glucosyl zeatin nucleotide were also detected.     

Phytohormones, Rhizobium mutants, and nodulation in legumes : v. Cytokinin metabolism in effective and ineffective pea root nodules

[³H]Zeatin riboside was supplied to intact pea (Pisum sativum) plants either onto the leaves or onto the root nodules. When applied directly to nodules, approximately 70% of recovered radioactivity remained in the nodules, approximately 15% was detected in the root system, and 15% was in the shoot. However, when supplied to the leaves, little ³H was transported, with approximately 0.05% of recovered radioactivity being found in the root system and nodules. On a fresh weight basis, nodules accumulated more ³H than the parent root. In both types of studies, metabolites with an intact zeatin moiety were detected in root nodules.

In all experiments, two-dimensional thin layer chromatography revealed that little ³H remained as zeatin riboside in root or nodule tissue at the end of the labeling period. Nodules metabolized [³H]zeatin riboside to the following cytokinins/cytokinin metabolites: zeatin, adenosine, adenine, the O-glucosides of zeatin and zeatin riboside, lupinic acid, nucleotides of adenine and zeatin, and the dihydro derivatives of many of these compounds.

Although a few small differences were observed, there were no major differences between root and nodule tissue in their metabolism of [³H] zeatin riboside. Furthermore, any differences between effective and ineffective nodules were generally minor.

     

Cytokinin Biochemistry in Relation to Leaf Senescence: IV. Cytokinin Metabolism in Soybean Explants

When [³H]dihydrozeatin riboside and [³H]zeatin riboside were supplied to soybean (Glycine max L.) explants (comprising one leaf, associated pods, and subtending stem) via the xylem at mid to late podfill, 0.1% of the supplied ³H was extracted from the seeds. The distribution of ³H in the explants was similar to that bound previously following uptake of [³H]zeatin riboside at earlier stages of pod development. Metabolites formed in the explants from ³H-labeled zeatin, zeatin riboside, and dihydrozeatin riboside were identified and related to the endogenous cytokinins shown to be present. When zeatin riboside and zeatin were supplied for 1 hour, zeatin nucleotide was the principal metabolite formed and this appeared to be the precursor of the other metabolites detected subsequently. Explants supplied with zeatin riboside or dihydrozeatin riboside for 1 hour, and then transferred to water for 20 to 24 hours, yielded leaf blades in which the main metabolites were O-glucosyldihydrozeatin, adenosine, and adenine. The metabolism of zeatin riboside in blades of explants at pre-podfill, early podfill, and mid to late podfill did not differ appreciably. The results are discussed in relation to leaf senescence and seed development.     

Ontogenetic variation of four cytokinins in soybean root pressure exudate

Cytokinins exported from the root may be involved in the correlative control of plant development. To test this hypothesis in soybean ((Glycine max [L.] Merr. cv. McCall, cv Chippewa 64, and cv Hodgson 78), cytokinins were intercepted en route from the root to the shoot by collecting root pressure exudate from detopped roots. The quantities of four cytokinins in the exudate were studied throughout the development of plants grown in the field and in controlled environment chambers. Zeatin, zeatin riboside, and their dihydro derivatives, dihydrozeatin and dihydrozeatin riboside, were isolated and quantitated using high-performance liquid chromatography.

Cytokinin fluxes (pmoles per plant per hour) were independent of exudate flux (grams per plant per hour). All fluxes are averages for a 6- or 8-h collection period. The ribosides accounted for the majority of the observed cytokinin transport. The fluxes of zeatin riboside and dihydrozeatin riboside increased from low levels during vegetative growth to maxima during late flowering or early pod formation. Before the seeds began rapid dry matter accumulation, zeatin riboside and dihydrozeatin riboside fluxes decreased and remained at low levels through maturation. The fluxes of zeatin and dihydrozeatin were low throughout development.

No correlation was found between cytokinin fluxes and nodule dry weight or specific nodule activity (acetylene reduction).

The timing of distinct peaks in zeatin riboside and dihydrozeatin riboside fluxes during flowering or pod formation suggests that cytokinins exported from the root may function in the regulation of reproductive growth in soybean.

     

Zeatin Glycosylation Enzymes in Phaseolus: Isolation of O-Glucosyltransferase from P. lunatus and Comparison to O-Xylosyltransferase from P. vulgaris

An enzyme catalyzing the formation of O-glucosylzeatin in immature embryos of Phaseolus lunatus was purified 2500-fold using ammonium sulfate precipitation followed by affinity and anion exchange chromatography. The enzyme uses trans-zeatin as substrate (K_m 28 micromolar) but not cis-zeatin, ribosylzeatin, or dihydrozeatin. Both UDP-glucose and UDP-xylose can serve as glycosyl donors, with K_ms of 0.2 and 2.7 millimolar, respectively, for the formation of O-glucosylzeatin and O-xylosylzeatin. In comparison, the UDPxylose-zeatin:O-xylosyltransferase (JE Turner, DWS Mok, MC Mok, G Shaw [1987] Proc Natl Acad Sci USA 84: 3714-3717) isolated by the same procedures from P. vulgaris embryos uses only UDP-xylose as donor substrate and the K_ms for both zeatin and UDP-xylose are much lower (2 and 3 micromolar, respectively). The chromatographic behavior on affinity columns and molecular weights (approximate M_r 44,000 daltons) of the two enzymes are similar. Results from substrate competition experiments and enzyme separation by anion exchange HPLC indicate a single, distinct, zeatin O-glycosylation enzyme occurs in embryos of each of these Phaseolus species.     

Phytohormones,Rhizobium mutants,and nodulation in legumes. VI. Metabolism of zeatin riboside applied via the tips of nodulated pea roots

[³H]zeatin riboside was supplied in physiological quantities to pea (Pisum sativum L. cv Greenfeast) plants by replacing the root tip with a small vial containing [³H]zeatin riboside, to simulate the normal supply of cytokinin. Radioactivity was transported to the root nodules. Analysis by two-dimensional thin layer chromatography revealed that little³H remained as zeatin riboside in root or nodule tissue at the end of the labeling period (2, 5, or 8 d) and suggested that the following compounds were metabolites of [³H]zeatin riboside: zeatin, adenosine, adenine, the O-glucosides of zeatin and zeatin riboside, nucleotides of adenine and zeatin, and the dihydro-derivatives of many of these compounds. The O-glucosides (and in particular, O-β-D-glucopyranosyl-9-β-D-ribofuranosylzeatin) appeared to be more prominent metabolites in the effective nodules formed by strain ANU897 than in the ineffective nodules produced by strain ANU203. However, no other appreciable differences were detected between effective and ineffective nodules in their metabolism of zeatin riboside. There were few marked differences between root and nodule tissue; however, in some experiments, the nodules contained a higher proportion of O-glucoside metabolites, and generally root tissue contained a greater proportion of zeatin and/or dihydro-zeatin, zeatin riboside and/or dihydrozeatin riboside, adenine and the nucleotides of zeatin and adenine, as metabolites.     

Cytokinin metabolism in nondividing and auxin-induced dividing explants ofHelianthus tuberosus L. Tuber tissue

Aqueous solutions of auxin (indole-3-acetic acid,α-naphthalene acetic acid, or 2,4-dichlorophenoxyacetic acid) were active in inducing DNA synthesis and mitosis in prewashed tissue explants of mature Jerusalem artichoke tubers. Explants did not respond in this way to aqueous solutions of cytokinin (zeatin, zeatin riboside, 6-benzylaminopurine, or kinetin). The metabolism of [8-³H]zeatin riboside (ZR) was studied in non-dividing and auxin-induced synchronously dividing explants over the first 36 h of culture. ZR was taken up rapidly and to the same extent by both tissues. Sequential analysis of tissue extracts by thin-layer and high-performance-liquid chromatography identified zeatin nucleotide(s) (ZN), O-glucosyl zeatin riboside (OGZR), adenosine, and adenine nucleotide(s) (AN) as the principal metabolites in both tissues. The proportion of radio-activity due to ZR declined steadily and OGZR accumulated steadily at similar rates in both tissues. ZN was the major metabolite in both tissues at 12 h; thereafter ZN continued to accumulate in nondividing tissue, but its level declined in dividing tissue, and a corresponding increase in the levels of AN and adenosine was observed. These treatment differences in cytokinin metabolism were apparent at least 6 h before the onset of mitosis.     

Role of catalase,H2O2 and phenolics in resistance of pigeonpea towards Helicoverpa armigera (Hubner)

Rapid generation of superoxide radicals and accumulation of H₂O₂ is a characteristic early response of plants following perception of insect herbivory signals. Induction of oxidative burst on account of herbivory triggers various defense mechanisms in plants. Response of superoxide and H₂O₂-metabolizing enzymes and secondary metabolites in nine pigeonpea genotypes to Helicoverpa armigera feeding was investigated. Out of nine, four genotypes were found to be moderately resistant, three were intermediate and two were moderately susceptible. In general, H. armigera infestation resulted in increase in superoxide dismutase activity, H₂O₂ and phenolics content and decrease in catalase (CAT) activity in leaves, developing seeds and pod wall of pigeonpea genotypes. Peroxidase activity was found only in leaves. Among genotypes, the increase in phenolic constituents was found greater in moderately resistant genotypes than in moderately susceptible genotypes; this might determine their contribution in providing resistance to genotypes against H. armigera infestation. The capability of moderately resistant genotypes to maintain relatively lower H₂O₂ content and higher CAT activity in pod wall and developing seeds also appeared to determine resistance of genotypes towards H. armigera. Expression of resistance to H. armigera was found to be associated with a negative correlation of H₂O₂-metabolizing enzymes and phenolics with pod damage as well as with negative association between CAT activity and H₂O₂ content. A positive correlation found between H₂O₂ content and pod damage suggested the accumulation of H₂O₂ in response to pod borer attack. In addition, correlation analysis also revealed a positive association between CAT, phenolic compounds and DPPH radical scavenging activity following pod borer attack; this indicated their contribution in resistance mechanisms against H. armigera herbivory.     

Movement to bark and metabolism of xylem cytokinins in stems of Lupinus angustifolius

Following uptake of [(3)H]zeatin riboside and [(3)H]dihydrozeatin riboside by girdled lupin (Lupinus angustifolius L.) stems via the transpiration stream, rapid lateral movement of the radioactivity from xylem to bark was observed. Short-term studies with intact stems, and other studies with excised stem tissues, revealed that the ribosides and/or the corresponding nucleotides were the cytokinin forms which actually moved into the bark tissues. Relative to cytokinin metabolism in xylem plus pith, metabolism in bark was both more rapid and more complex. Riboside cleavage and formation of the O-acetylzeatin and O-acetyldihydrozeatin ribosides and nucleotides were almost completely confined to bark tissues. Exogenous (3)H-labelled O-acetylzeatin riboside was converted to zeatin riboside in bark tissue, but the presence of the acetyl group suppressed degradation to adenine metabolites. The sequestration and modification of xylem cytokinins by stem tissues probably contributes significantly to the cytokinin status of the shoot. New cytokinins identified by mass spectrometry in lupin were: O-acetyldihydrozeatin 9-riboside, a metabolite of exogenous dihydrozeatin riboside in stem bark; O-methylzeatin nucleotide and O-methyldihydrozeatin 9-riboside, metabolites of endogenous cytokinins in stem bark; O-methylzeatin nucleotide and O-methylzeatin 9-riboside, metabolites of exogenous zeatin riboside in excised pod walls.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> plantlet production of the endangered <Emphasis Type="Italic">Pinguicula vulgaris</Emphasis>

This study describes the development of a micropropagation protocol for Pinguicula vulgaris using cultures initiated from in vitro produced seedlings. P. vulgaris is a carnivorous plant with a northern, disjunctly circumpolar distribution and specific habitat requirements, and is hence becoming increasingly rare. Shoot proliferation was significantly influenced by Murashige and Skoog (MS) macronutrient concentration, showing higher proliferation rates in 1/4MS, but was not affected by the addition of 0.1 mg/L 6-benzyladenine (BA) or zeatin (Zea). The best medium for propagating P. vulgaris was plant growth regulator (PGR) free ¼MS. An average of 7.62 new shoots per initial explant could be obtained after 8 weeks of culture, of which over 79% produced roots during proliferation. Moreover, rooting percentages of 100% were obtained for the initial explants in all the tested media, including media without PGRs. The plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development.     

Cytokinin metabolism and the modulation of cytokinin activity in radish

The metabolism of zeatin, N⁶-(Δ²-isopentenyl) adenine, dihydrozeatin, zeatin-O-glucoside and dihydrozeatin-O-glucoside has been studied using derooted radish seedlings. The metabolites were identified by UV and GC/MS. The patterns of metabolism are compared and provide evidence that the O-glucosyl conjugates may be storage forms of the cytokinins.     

Short-term UV-B radiation effects on morphology,physiological traits and accumulation of bioactive compounds in Prunella vulgaris L.

Prunella vulgaris plants (full-bloom stage) were subjected to short-term (15 days) UV-B radiation in a growth chamber. UV-B radiation was effective at enhancing morphological and biomass characteristics and decreasing chlorophyll contents of P. vulgaris. Treatment of P. vulgaris with artificial UV-B radiation significantly increased peroxidase (POD), superoxide dismutase (SOD) and glutathione (GSH) activities compared to the control treatment. UV-B radiation significantly increased the levels of hydrogen peroxide (H₂O₂), malondialdehyde (MDA) and proline in leaves of P. vulgaris compared to those of control plants. In addition, the contents of total flavonoids, rosmarinic acid, caffeic acid and hyperoside significantly increased under UV-B radiation. The total phenolic levels also increased under UV-B treatment. These results demonstrated that short-term UV-B radiation can enhance production of secondary metabolites in P. vulgaris, resulting in increased spica yield compared to that of control plants.     

Evidence for Host Genome Involvement in Cytokinin Metabolism by Male and Female Cells of Mercurialis annua Transformed by Strain 15,955 of Agrobacterium tumefaciens

When male and female individuals of a dioecious species Mercurialis annua L. were inoculated with the same strain of Agrobacterium tumefaciens (15,955), the corresponding tumor tissues of each sex clearly differed in their endogenous cytokinin content; only the male tumors had a morphogenetic feminizing effect on male flowers.

In male tumor tissues, zeatin (Z) in higher quantity than ribosyl-zeatin (RZ) became the major metabolite in contrast with the general situation for crown-galls; the female tumor tissues were characterized by an increase of total endogenous cytokinins and by the appearance of some specific metabolites such as a methyl-thio-Z and several glycosylated Z derivatives that had not been detected in healthy apices.

In both male and female tumor tissues, the cis form of RZ, present in healthy apices as 30% of trans-RZ form, was no longer detectable.

Quantitative and qualitative differences characterize male and female tumor tissues (host genes expression) but since differences also appeared between healthy male and female apices and their corresponding tumor tissues (TDNA gene expression), it can be tentatively concluded that a complex interaction between host cytokinin genes and those of TDNA control the endogenous metabolism of tumor tissues.

     

Cytokinin autonomy in tissue cultures of phaseolus: a genotype-specific and heritable trait

Intra- and interspecific differences in cytokinin requirement were detected in callus cultures of Phaseolus vulgaris L. and P. lunatus L. Of the ten genotypes of P. vulgaris tested in the present study, one required cytokinin for callus growth, six exhibited some to moderate growth on cytokinin-free medium, and the remaining three grew uniformly in the absence of cytokinin. In contrast, six of the P. lunatus genotypes were strictly cytokinin-dependent, while four genotypes displayed irregular amount of callus growth on cytokinin-free medium. The genotype-specific behavior of Phaseolus callus tissues was independent of the tissue of origin and the time in culture. The inheritance of the cytokinin requirement of Phaseolus tissue cultures was studied in hybrid tissues resulting from crosses between a strictly cytokinin-dependent genotype (P.I. 200960) and two independent genotypes (cv. G 50 and P.I. 286303) of P. vulgaris. Fresh weights of hybrid tissues on cytokinin-free medium were intermediate between and significantly different from the parental tissues. No differences were found between reciprocal hybrids. These results suggest that cytokinin autonomy in tissue cultures of P. vulgaris is a genetic trait under nuclear control. Both parental and intermediate phenotypes were recovered in the F₂ progeny. The frequency distribution of cytokinin-dependent progeny in F₂ and backcross populations indicates that the cytokinin requirement of P. vulgaris callus tissue may be regulated by one set of alleles.     

Cytokinin structure-activity relationships and the metabolism of N-(delta-isopentenyl)adenosine-8-C in phaseolus callus tissues

The activities of the free base and ribonucleoside forms of cytokinins bearing saturated and unsaturated N⁶-isoprenoid side chains have been examined in callus cultures derived from Phaseolus vulgaris cv. Great Northern, P. lunatus cv. Kingston, and the interspecific hybrid Great Northern × Kingston. In callus of cv. Great Northern, cytokinins bearing saturated side chains (N⁶-isopentyladenine, N⁶-isopentyladenosine, dihydrozeatin, and ribosyldihydrozeatin) were always more active than the corresponding unsaturated analogs (N⁶-[Δ²-isopentenyl]adenine, N⁶-[Δ²-isopentenyl]adenosine, zeatin, and ribosylzeatin). In callus of cv. Kinston, the cytokinins bearing unsaturated side chains were either more active or equally as active as the saturated compounds. These differences in cytokinin structure-activity relationships were correlated with differences in the metabolism of ¹⁴C-N⁶-(Δ²-isopentenyl)adenosine. In Great Northern tissues, this cytokinin was rapidly degraded to adenosine; in Kingston tissues, the major metabolite was the corresponding nucleotide. The growth responses of callus of the interspecific hybrid were intermediate between the parental tissues, and the metabolism of ¹⁴C-N⁶-(Δ²-isopentenyl)adenosine by the hybrid callus exhibited characteristics of both parental tissues. The results are consistent with the hypothesis that the weak activity of cytokinins with unsaturated side chains in promoting the growth of Great Northern callus is due to the rapid conversion of these cytokinins to inactive metabolites.