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.     

Cytokinin Oxidase from Phaseolus vulgaris Callus Cultures : Affinity for Concanavalin

Cytokinin oxidase activity from Phaseolus vulgaris cv Great Northern callus cultures exhibited affinity for the lectin concanavalin A. Over 80% of the activity extracted from the callus tissue bound to a concanavalin A-Sepharose 4B column. The bound activity was eluted from the column by the addition of methylmannose to the eluting buffer. On the basis of this result, it appears that most of the cyokinin oxidase activity present in Great Northern callus cultures exists in the form of a glycoprotein. The apparent pI of this enzyme, as estimated by chromatofocusing, is approximately 5.0.     

Cytokinin Oxidase from Phaseolus vulgaris Callus Tissues : Enhanced in Vitro Activity of the Enzyme in the Presence of Copper-Imidazole Complexes

The effects of metal ions on cytokinin oxidase activity extracted from callus tissues of Phaseolus vulgaris L. cv Great Northern have been examined using an assay based on the oxidation of N⁶-(Δ²-isopentenyl)-adenine-2,8-³H (i⁶ Ade) to adenine (Ade). The addition of cupric ions to reaction mixtures containing imidazole buffer markedly enhanced cytokinin oxidase activity. In the presence of optimal concentrations of copper and imidazole, cytokinin oxidase activity was stimulated more than 20-fold. The effect was enzyme dependent, specific for copper, and observed only in the presence of imidazole. The substrate specificity of the copper-imidazole enhanced reaction, as judged by substrate competition tests, was the same as that observed in the absence of copper and imidazole. Similarly, in tests involving DEAE-cellulose chromatography, elution profiles of cytokinin oxidase activity determined using a copper-imidazole enhanced assay were identical to those obtained using an assay without copper and imidazole. On the basis of these results, the addition of copper and imidazole to reaction mixtures used to assay for cytokinin oxidase activity is judged to provide a reliable and specific assay of greatly enhanced sensitivity for the enzyme. The mechanism by which copper and imidazole enhance cytokinin oxidase activity is not certain, but the reaction catalyzed by the enzyme was not inhibited by anaerobic conditions when these reagents were present. This observation suggests that copper-imidazole complexes are substituting for oxygen in the reaction mechanism by which cytokinin oxidase effects cleavage of the N⁶-side chain of i⁶Ade.     

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.     

Regulation of Cytokinin Oxidase Activity in Callus Tissues of Phaseolus vulgaris L. cv Great Northern

The regulation of cytokinin oxidase activity in callus tissues of Phaseolus vulgaris L. cv Great Northern has been examined using an assay based on the oxidation of N⁶-(Δ²-isopentenyl)adenine-8-¹⁴C (i⁶ Ade-8-¹⁴C) to adenine. Solutions of exogenous cytokinins applied directly to the surface of the callus tissues induced relatively rapid increases in cytokinin oxidase activity. The increase in activity was detectable after 1 hour and continued for about 8 hours, reaching values two- to three-fold higher than the controls. The cytokinin-induced increase in cytokinin oxidase activity was inhibited in tissues pretreated with cordycepin or cycloheximide, suggesting that RNA and protein synthesis may be required for the response. Rifampicin and chloramphenicol, at concentrations that inhibited the growth of Great Northern callus tissues, were ineffective in inhibiting the increase in activity. All cytokinin-active compounds tested, including both substrates and nonsubstrates of cytokinin oxidase, were effective in inducing elevated levels of the enzyme in Great Northern callus tissue. The cytokinin-active urea derivative, Thidiazuron, was as effective as any adenine derivative in inducing this response. The addition of Thidiazuron to the reaction volumes used to assay cytokinin oxidase activity resulted in a marked inhibition of the degradation of the labeled i⁶ Ade-8-¹⁴C substrate. On the basis of this result, it is possible that Thidiazuron may serve as a substrate for cytokinin oxidase, but other mechanisms of inhibition have not yet been excluded.     

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 activity of N-phenyl-N′-1, 2,3-thiadiazol-5-ylurea (thidiazuron)

The plant growth regulator N-phenyl-N′-1, 2, 3-thiadiazol-5-ylurea (Thidiazuron) displayed high activity in promoting the growth of cytokinin-dependent callus cultures of Phaseolus lunatus cv. Kingston. The cytokinin activity of Thidiazuron was similar to that of the highly active N-phenyl-N′-4-pyridylurea derivatives and to the most active cytokinins of the adenine type. Replacement of the phenyl ring of Thidiazuron with other ring structures resulted in a decrease in cytokinin activity.     

Differential cytokinin structure-activity relationships in phaseolus

The activities of eight cytokinins in promoting callus growth were tested in two Phaseolus genotypes, P. vulgaris L. var. Great Northern, and P. lunatus L. var. Kingston. The structural feature which contributes to the major genotypic difference in cytokinin structure-activity relationships is the presence or absence of a double bond at the 2,3-position of the isoprenoid N⁶ side chain. In Kingston, trans-zeatin was 3-fold more active than dihydrozeatin and 30-fold more active than cis-zeatin. The activities of N⁶-(Δ²-isopentenyl)adenine and N⁶-isopentyladenine were nearly the same. In Great Northern, however, dihydrozeatin was at least 30-fold more active than both trans-zeatin and cis-zeatin, and N⁶-isopentyladenine was 100-fold more active than N⁶-(Δ²-isopentenyl)adenine. The results suggest the possibility of employing cytokinin structure-activity relationships in distinguishing genotypic differences in cytokinin function and metabolism.     

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     

The influence of aluminium availability on phosphate uptake in Phaseolus vulgaris L. and Phaseolus lunatus L.

Aluminium toxicity is one of the major limiting factors of crop productivity on acid soils. High levels of available aluminium in soil may induce phosphorus deficiency in plants. This study investigates the influence of Aluminium (Al) on the phosphate (P_i) uptake of two Phaseolus species, Phaseolus vulgaris L. var. Red Kidney and Phaseolus lunatus L. The two bean species were treated first with solutions of Al at different concentrations (0, 25, 50 and 100 μM, pH 4.50) and second with solutions of P_i (150 μM) at pH 4.50. The higher the Al concentration the higher the Al concentration sorbed but P. vulgaris L var. Red Kidney adsorbed significantly more Al than P. lunatus L. Both species released organic acids: P. vulgaris L var. Red Kidney released fumaric acid and P. lunatus L. fumaric and oxalic acids which could have hindered further Al uptake.The two bean species showed a sigmoid P_i uptake trend but with two different mechanisms. P. vulgaris L var. Red Kidney showed a starting point of 3 h whereas P. lunatus L. adsorbed P_i immediately within the first minutes. In addition, P. vulgaris L var. Red Kidney presented significantly higher P_i uptake (higher uptake rate ‘k’ and higher maximum adsorption ‘a’ of the kinetic uptake model). The Al treatments did not significantly influence P_i uptake. Results suggest that P. lunatus L. might adopt an external Al detoxification mechanism by the release of oxalic acid. P. vulgaris L var. Red Kidney on the other hand seemed to adopt an internal detoxification mechanism even if the Al sorbed is poorly translocated into the shoots. More detailed studies will be necessary to better define Al tolerance and/or resistance of Phaseolus spp.     

Hormonal Regulation of the Lectin Biosynthesis in Callus Culture of the Phaseolus vulgaris Plant

Callus cultures established from Phaseolus vulgaris seedlings were used to investigate hormonal influence on lectin biosynthesis. The plant tissue cultures were initiated using defined levels of both a cytokinin (kinetin) and an auxin (2,4-dichlorophenoxyacetic acid) and were then transferred to media containing different amounts of these hormones. The lectin content of each callus culture was determined using an enzyme immunoassay specific for the seed lectin of the P. vulgaris plant. The lectin biosynthesis was directly affected by the levels of auxin and cytokinin in the culture media and no lectin was detected in hormone-free medium. This enabled us to compose culture media yielding a maximal or minimal lectin content of the callus cultures, illustrating the ability to induce an enhancement or suppression of the in vitro lectin biosynthesis. The lectin level of callus tissue during the growth cycle of a culture was, furthermore, related to the cellular growth rate which might indicate an involvement of the lectin in cellular events during rapid cell division.     

The metabolism of [14C]-tMdiaziiroii in callus tissues of Phaseolus lunatus

The metabolism of [¹⁴C]-thidiazuron (N-phenyl-N'-l,2,3-tbiadiazol-5-ylurea), a compound with extremely high cytokinin activity, was examined in callus cultures of Phaseolus lunatus L. cv . Kingston. No appreciable metabolism of the compound was detected in short-term studies (up to 48 h). When tissues were grown on medium containing [¹⁴C]-thidiazuron for longer periods (12 to 33 days), several major metabolites were isolated. The array of radioactive metabolites was the same using [¹⁴C]-thidiazuron with the label in the phenyl ring, the urea bridge or the thiadiazol ring, indicating no degradation of the thidiazuron molecule. Enzyme digestion followed by HPLC and TLC analyses was used to identify major metabolites. α-Gtacosidase degraded two of the metabolites to thidiazuron and a third was converted to N-p-hydroxyphenyl-N²-l,2,3-thiadiazol-5-ylurea. Limited tests of the O-glycosyl derivatives for cytokinin activity indicated that they are much less active than the parent compound. Bioassays involving hydroxylated derivatives of thidiazuron indicated that the p-hydroxyphenyl derivative was 10000 limes less active than the parent compound, and the oetho - and mete -derivatives had 1000 and 100 times lower activity, respectively, than thidiazuron. These results suggest that thidiazuron itself is the active compound and that metabolism of thidiazuron in Phaseolus tissues only involves modification of the intact structure, yielding metabolites that are less cytokinin-active.     

Effects of Thidiazuron on Cytokinin Autonomy and the Metabolism of N-(Delta-Isopentenyl)[8-C]Adenosine in Callus Tissues of Phaseolus lunatus L

The effects of a highly cytokinin-active urea derivative, N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (Thidiazuron), and zeatin on cytokinin-autonomous growth and the metabolism of N⁶-(Δ²-isopentenyl)[8-¹⁴C]adenosine ([¹⁴C]i⁶ Ado) were examined in callus tissues of two Phaseolus lunatus genotypes, cv Jackson Wonder and P.I. 260415. Tissues of cv Jackson Wonder maintained on any concentration of Thidiazuron became cytokinin autonomous, whereas only tissues exposed to suboptimal concentrations of zeatin displayed cytokinin-autonomous growth. Tissues of P.I. 260415 remained cytokinin dependent under all these conditions. The metabolism of [¹⁴C]i⁶ Ado was similar for the two genotypes, but differed with the medium used. [¹⁴C]i⁶ Ado was rapidly converted to N⁶-(Δ²-isopentenyl)[8-¹⁴C]adenosine 5′-P ([¹⁴C]i⁶ AMP) by tissues grown on zeatin-containing medium, whereas only traces of the nucleotide were formed in tissues grown on medium with Thidiazuron. Incubation with [¹⁴C] i⁶ AMP of tissues grown in the presence of Thidiazuron resulted in rapid conversion to [¹⁴C]i⁶ Ado, while [¹⁴C]i⁶ AMP persisted in tissues maintained on zeatin. Thus, Thidiazuron appears to stimulate enzyme activity converting the ribonucleotide to ribonucleoside. Although the cytokininactive phenylureas and adenine derivatives differ in their effects on cytokinin autonomy as well as nucleotide formation, the two types of effects do not seem to be related.     

Regeneration of different cultivars of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) via indirect organogenesis

A protocol for in vitro regeneration via indirect organogenesis for Phaseolus vulgaris cv. Negro Jamapa was established. The explants used were apical meristems and cotyledonary nodes dissected from the embryonic axes of germinating seeds. Several auxin/cytokinin combinations were tested for callus induction. The best callus production was obtained with medium containing 1.5 μM 2,4-dichlorophenoxyacetic acid. After 2 weeks of growth calli were transferred to shooting medium containing 22.2 μM 6-benzylaminopurine. Shoots regenerated with a frequency of approximately 0.5 shoots per callus, and upon transfer to rooting medium these shoots produced roots with 100% efficiency. Histological analyses of the regeneration process confirmed the indirect organogenesis pattern. Greenhouse grown regenerated plants showed normal development and were fertile. The protocol was reproducible for other nine P. vulgaris cultivars tested, suggesting a genotype independent procedure.     

Abscisic Aldehyde Is an Intermediate in the Enzymatic Conversion of Xanthoxin to Abscisic Acid in Phaseolus vulgaris L. Leaves

The enzymatic conversion of xanthoxin to abscisic acid by cell-free extracts of Phaseolus vulgaris L. leaves has been found to be a two-step reaction catalyzed by two different enzymes. Xanthoxin was first converted to abscisic aldehyde followed by conversion of the latter to abscisic acid. The enzyme activity catalyzing the synthesis of abscisic aldehyde from xanthoxin (xanthoxin oxidase) was present in cell-free leaf extracts from both wild type and the abscisic acid-deficient molybdopterin cofactor mutant, Az34 (nar2a) of Hordeum vulgare L. However, the enzyme activity catalyzing the synthesis of abscisic acid from abscisic aldehyde (abscisic aldehyde oxidase) was present only in extracts of the wild type and no activity could be detected in either turgid or water stressed leaf extracts of the Az34 mutant. Furthermore, the wilty tomato mutants, sitiens and flacca, which do not accumulate abscisic acid in response to water stress, have been shown to lack abscisic aldehyde oxidase activity. When this enzyme fraction was isolated from leaf extracts of P. vulgaris L. and added to extracts prepared from sitiens and flacca, xanthoxin was converted to abscisic acid. Abscisic aldehyde oxidase has been purified about 145-fold from P. vulgaris L. leaves. It exhibited optimum catalytic activity at pH 7.25 in potassium phosphate buffer.     

Isolation and Identification of the Precursor of Ethane in Phaseolus vulgaris L

Ethane production by homogenates of Phaseolus vulgaris L. cv. Harvester was studied. The precursor of ethane was identified as linolenic acid. The liberation of ethane was optimum at pH 4.2 and was highest from homogenates of leaves and apical buds. When roots were homogenized in linolenic acid solution, ethane and ethylene production were stimulated. In corn root homogenates, ethylene biosynthesis was stimulated nearly 8-fold by linolenic acid. The enzyme responsible for ethane production from oat root homogenates was soluble and had a high molecular weight.     

Isozymes of Glutamine Synthetase in Phaseolus vulgaris L. and Phaseolus lunatus L. Root Nodules

The glutamine synthetase (GS) isozymes in the plant fraction of nodule extracts from 62 cultivars of Phaseolus vulgaris L. and one cultivar of Phaseolus lunatus L. were analyzed by polyacrylamide gel electrophoresis. All P. vulgaris nodule extracts displayed two GS activity bands: a nodule-specific band (GS_n1) and a band (GS_n2) similar to the single band (GS_r) present in root extracts. In nodule extracts of P. lunatus, the GS_n1 band was detected, but the GS_n2 band was barely detectable. In contrast to P. vulgaris, the GS_n2 band and the GS_r band of P. lunatus appeared to be different. The electrophoretic mobility of the GS_n1 band in P. vulgaris was governed by both the plant cultivar and the development stage of the nodule. In nodule extracts of P. vulgaris and P. lunatus, the zone of GS_n1 activity coincided with six to nine distinct protein bands as revealed after treatment of gels, which had previously been stained for GS activity, with Coomassie blue. All these protein bands were shown to consist of polypeptides of identical molecular weight (approximately 47,000 daltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results indicate that P. vulgaris continuously generates isozymes of GS_n1 of increasing electrophoretic mobility during the course of nodule development.     

Zeatin Metabolism in Fruits of Phaseolus: Comparison between Embryos, Seedcoat, and Pod Tissues

Zeatin metabolites were isolated from seedcoats and pod tissues of Phaseolus vulgaris and P. lunatus. The differences observed previously between P. vulgaris and P. lunatus embryos, i.e. the formation of O-ribosyl derivatives in the former and O-glucosyl derivatives in the latter, could also be detected in seedcoats, although the levels of these metabolites were much lower and there was a concomitant increase of breakdown products (adenine, adenosine and AMP). Inner pod wall tissues of both genotypes metabolized zeatin at a slow rate and the major metabolite was the mononucleotide of zeatin. The array of metabolites recovered was not influenced by the extraction method (cold ethanol or modified Bieleski solution).