Production of cytokinin-like substances by mycorrhizal fungi of pine (Pinus sylvestris L.) in cultures with and without metabolites of actinomycetes

Studies on the effect of post culture liquids of actinomycetes on cytokinin-like substances production by mycorrhizal fungi have revealed that actinomycete metabolites inhibited or stimulated the synthesis of these compounds. The results of chromatographic analyses suggest, that substances stimulating the soybean callus are likely to be: riboside 6 (gamma, gamma-dimethylallylamino) purine and riboside zeatin. Using gas chromatography it was confirmed that both substances are produced by Rhizopogon luteolus. Paxillus involutus synthesizes probably besides the two substances also zeatin, as appears from the data obtained by column chromatography.     

Pathogenesis of barley leaves by Helminthosporium teres I: Green island formation and the possible involvement of cytokinins

Infection sites/green islands were formed in host leaf tissue infected with drops of H. teres. They exhibited higher cytokinin-like activity, sugar and starch than their surrounding tissue and tissue under water drops. The cytokinin-like activity at the infection sites increased from 24 to 72 h of incubation. However, the cytokinin-like activity of the tissue surrounding the infection drops and the tissue under water drops fell from 24 to 72 h incubation. The culture filtrate extracts of the fungus also produced cytokinin-like activity which increased from 1 to 10 days incubation. Application of this culture filtrate extract evoked green island formation. Application of kinetin to host leaves duplicated the green island effect. Thin-layer chromatographic fractions of the tissue extracts and the culture filtrate extracts revealed that a major portion of cytokinin-like activity corresponded to zeatin and zeatin riboside. The presence of zeatin and zeatin riboside (both in tissue and culture filtrate extracts) was confirmed by high performance liquid chromatography. Increases in the amounts of cytokinin-like substances, particularly zeatin and zeatin riboside, attributed to pathogen influence are suggested to be involved in infection and pathogenicity of H. teres.     

Effect of pH on production of cytokinin-like substances by bacteria isolated from soil, rhizosphere and mycorrhizosphere of pine (Pinus silvestris L.)

Cytokinin-like substances were produced in higher amounts by soil isolates than by those isolated from the rhizosphere and mycorrhizosphere. However more bacterial strains isolated from the rhizosphere and mycorrhizosphere were capable of synthesizing cytokinin-like compounds. A distinct effect of pH on the production of these substances was found. It is suggested that zeatin and zeatin riboside are likely to be among the cytokinin-like substances detected.     

Comparison of cytokinin- and auxin-like activity in some commercially used seaweed extracts

Six commercially used seaweed extracts were tested for cytokinin- and auxin-like activity using the soybean callus bioassay and the mung bean rooting bioassay respectively. All the seaweed extracts tested showed cytokinin-like activity. Seamac caused the most callus growth with activity being concentrated at R _f 0.9 which co-chromatographed withiso-pentenyladenine and its riboside. The other five seaweed extracts yielded activity at R _f 0.7–0.8. This co-chromatographed with zeatin and zeatin riboside. All the seaweed extracts tested improved rooting of mung beans. Kelpak made fromEcklonia maxima (Osbeck) Papenf. gave the best rooting response.     

The occurrence of free and bound cytokinins in clubroots and Plasmodiophora brassicae infected turnip tissue cultures

This paper deals with the quantitative determination of free and bound cytokinins in clubroot tissue and in Plasmodiophora brassicae Woron, infected Brassica campestris L. callus tissue. The fractions were separated in a butanol soluble fraction containing the free cytokinins such as zeatin and zeatin riboside and a water soluble fraction containing the bound cytokinins. The butanol fraction was extensively purified and analysed by high pressure liquid chromatography (HPLC). The butanol fraction contained cytokinins which cochromatographed with zeatin and zeatin riboside and not with dihydrozeatin. Zeatin and zeatin riboside were quantitatively determined by HPLC. Recovery of the cytokinins varied between 30–50%. Clubs contained 50–160 ng zeatin and 210–300 ng zeatin riboside per g dry weight. Callus tissue contained 133 ng zeatin and 169 ng zeatin riboside per g dry weight. Clubs, callus as well as healthy tissue contain large amounts of bound cytokinins. Upon treatment of the water soluble fraction first with alkaline phosphatase and then with β-glucosidase biologically active fractions were found which coeluted with zeatin and zeatin riboside on Sephadex LH₂₀ in 20% ethanol. Evidence is presented for a novel cytokinin in the water soluble fraction which yields free zeatin and glucose-6-phosphate after treatment with β-glucosidase.     

Cytokinins in the Phloem Sap of White Lupin (Lupinus albus L.)

Cytokinin-like activity in samples of xylem and phloem sap collected from field-grown plants of white lupin (Lupinus albus L.) over a period of 9 to 24 weeks after sowing was measured using the soybean hypocotyl callus bioassay following paper chromatographic separation. The phloem sap was collected from shallow incisions made at the base of the stem, the base of the inflorescence (e.g. stem top), the petioles, and the base and tip of the fruit. Xylem sap was collected as root exudate from the stump of plants severed a few centimeters above ground level. Concentration of cytokinin-like substances was highest in phloem sap collected from the base of the inflorescence and showed an increase over the entire sampling period (from week 10 [61 nanogram zeatin equivalents] to week 24 [407 nanogram zeatin equivalents]). Concentrations in the xylem sap and in the other phloem saps were generally lower. Relatively high concentrations of cytokinin-like substances in petiole phloem sap (70 to 130 nanogram zeatin equivalents per milliliter) coincided in time with high concentrations in sap from the base of the inflorescence (see above). Concentrations in sap (phloem or xylem) from the base of the stem were very much lower. This finding is consistent with movement of cytokinins from leaves into the developing inflorescence and fruit, rather than direct input to the fruit from xylem sap. However, an earlier movement of cytokinins from roots into leaves via the xylem cannot be ruled out. Sap collected at an 18-week harvest was additionally separated by sequential C₁₈ reversed-phase high performance liquid chromatography → NH₂ normal phase high performance liquid chromatography, bioassayed, and then analyzed by electron impact gas chromatography-mass spectrometry. Identification of zeatin riboside and dihydrozeatin as two of the major cytokinins in combined sap samples was accomplished by gas chromatography-mass spectrometry-selected ion monitoring.     

A cytokinin complex from the developing fruits of Aegle marmelos

Following ion-exchange, Sephadex LH-20 and paper chromatography of the methanolic extracts of young, developing fruits of Aegle marmelos Correa, cytokinin-like activity in the soybean callus assay was detected in six fractions. Of the four butanol-soluble compounds, two were tentatively identified as zeatin and zeatin riboside, and the others as zeatin glucoside and zeatin riboside glucoside. The major cytokinino of the butanol-insoluble fraction is probably zeatin nucleotide. The levels of compounds with cytokinin-like activity were high during the early phase, and low in the subsequent period of fruit growth. The activity resembling that of cytokinin glucoside increased with maturation of the fruit. The content of free cytokinins in the fruits was more than that released from the tRNA.     

The possible involvement of cytokinins in the pathogenicity of Helminthosporium maydis

Green islands/infection sites recorded higher cytokinin activity than surrounding tissue as well as non-inoculated tissue. This activity in infected areas increased with time of incubation while in tissue surrounding the green islands and non-inoculated tissue, cytokinin activity decreased with time of incubation. The culture filtrate extracts of H. maydis had cytokinin activity which increased with growth of the fungus. Cytokinin activity of thin-layer Chromatographic fractions from tissue and culture filtrate extracts revealed that a major portion of the activity was confined to Rf zone 0.6 to 0.8 which co-chromatographed with zeatin and zeatin riboside. Presence of zeatin and zeatin riboside in tissue and culture filtrates was confirmed by high performance liquid chromatography. Cytokinin substances, such as zeatin and zeatin riboside, increase at infection sites with growth of the pathogen suggesting they may be involved in the pathogenicity of H. maydis on maize.     

Increase of endogenous zeatin riboside by introduction of the ipt gene in wild type and the lateral suppressor mutant of tomato

We studied axillary meristem formation of the lateral suppressor (ls) mutant of tomato after elevating the endogenous cytokinin levels through introduction of the isopentenyltransferase (ipt) gene from Agrobacterium tumefaciens. Growth and development of several transformants were examined during in vitro culture. Transformants exhibited phenotypes varying in severity and were divided into four classes. A number of the ipt transformants had a normal phenotype, as non-transformed plants. Others showed a mild to severe ‘cytokinin-like’ phenotype. Transformants with a mild phenotype exhibited reduced internode length and reduced root development. Transformants with a severe phenotype showed even shorter internodes, loss of apical dominance, reduction of leaf size, production of callus at the basis of the shoots and absence of root development or development of green non-branching roots. The severity of the phenotype correlated well with the level of ipt gene expression, as measured by northern analysis. Transformants with a severe phenotype also exhibited increased levels of zeatin riboside, but zeatin levels were not elevated. The increase in endogenous zeatin riboside levels in the ls mutant did not restore axillary meristem formation, but sometimes bulbous structures were formed in the initially ‘empty’ leaf axils. Several adventitious meristems and shoots developed from below the surface of these structures. It is concluded that a reduced level of cytokinins in the ls mutant shoots is not responsible for the absence of axillary meristem formation.     

Occurrence of plant hormone (cytokinin)-producing bacteria in the sea

Plant hormones, which are considered to be cytokinins, were detected in the culture media of marine bacteria using the Amaranthus bioassay method. The proportion of cytokinin-producing bacteria to total microbes tested was higher in sediments (45–55%) than in seawater (5–15%). The amount of cytokinin-like substances in the culture media was estimated as 0.05–0.30 μg of zeatin equivalents/l. Thin layer chromatography analysis using the soybean callus bioassay method suggested that the active substance produced by one of these bacteria was isopentenyladenine or its riboside. Taxonomic examination of the cytokinin-producing bacteria showed that the production of the hormone was not specific to any genus. A possible role of cytokinins produced by sediment bacteria on the development of red tide is discussed.     

Cytokinins in Gladiolus (Gladiolus grandiflorus) Corms

Ten cytokinin-like substances, termed X₁, X₂, X₃, X_4a, X_4b,X_5a, X_5b, X₆, X₇, and X₈, active in the soya bean hypocotyltest, were detected in gladiolus corms. The factors X_4a, X_5aand X₄ were tentatively identified as zeatin (Z), isopentenyladenosine (iPA) and isopentenyl adenine (iP), respectively,based on their behaviour in Sephadex LH-20 column chromatography,paper chromatography and high pressure liquid chromatography.Factor X₃, which behaved like zeatin riboside (ZR) in the abovesystems could be ZR and/or dihydrozeatin riboside (DHZR). Thebehaviour in Sephadex and ion-exchange column chromatographysuggested that X₁ and X₂ may be cytokinin glucosides and X₅a cytokinin nucleotide or a cytokinin conjugate similar to lupinicacid. The total cytokinin content and the concentration of Z,ZR/DHZR and iPA were higher in non-dormant than in dormant corms.The concentrations of X₁ and X₂ were higher in dormant corms. Gladiolus grandiflorus, gladiolus, dormancy, cytokinins, Sephadex column chromatography, high pressure liquid chromatography     

Hormones in Plants Bearing Nitrogen-fixing Root Nodules: Cytokinin Transport from the Root Nodules of Alnus glutinosa (L.) Gaertn.

The movement and metabolism of [8-¹⁴C]zeatin applied to theroot nodules of Alnus glutinosa (L.) Gaertn, was investigated.Twenty-four hours after the start of uptake, zeatin and a numberof its metabolites were detected in all parts of the plant.The major radioactive compounds present in a cationic fractionof different plant parts at this time co-chromatographed onSephadex LH20 with zeatin (in nodules, stems, and leaves) andwith zeatin riboside (in roots, stems, and buds). In the roots,in addition to the peak co-chromatographing with zeatin riboside,there was also a prominent unidentified polar peak. The presence of zeatin and zeatin riboside in the stems andleaves was indicated also by chromatographic behaviour in othersystems, effects of permanganate oxidation, and cocrystallisationwith the authentic unlabelled compounds. Biological activitywas exhibited by both peaks in the soybean callus bioassay.Other metabolites in the shoot, possibly active as cytokinins,had the characteristics of dihydrozeatin, zeatin or dihydrozeatin-5'-nucleotide(s),and zeatin or dihydrozeatin glucosides. The gradual disappearancewith time of zeatin and its riboside from the shoot was accompaniedby an increase in the proportion of more polar metabolites. These results are discussed in relation to the possible exportof endogenous cytokinins by the nodules.     

The effect of aluminum on cytokinins in the mycelia of Amanita muscaria

High performance liquid chromatography analysis of immunoaffinity-purified extracts of mycelia of Amanita muscaria, and the Amaranthus bioassay of the eluted fractions, revealed the following seven cytokinins: zeatin, zeatin riboside, zeatin N-9-glucoside, dihydrozeatin, dihydrozeatin riboside, isopentenyl adenine, and isopentenyl adenosine. The decreased growth of aluminum-treated mycelia correlated with a 35% decrease in the total amount of the cytokinins. Among individual cytokinins, zeatin was the most affected, exhibiting a reduction of about 90%. The results are compared with previous investigations of aluminum effects on cytokinins in the mycelia of Lactarius piperatus, whose growth is stimulated by aluminum.Abbreviations ZR zeatin riboside - iPA isopentenyl adenosine - Z zeatin - DHZ dihydrozeatin - iP isopentenyl adenine - DHZR dihydrozeatin riboside - Z-9G zeatin N-9-glucoside - iP-9G isopentenyl N-9-glucoside - HPLC high performance liquid chromatography - DHZRMP dihydrozeatin riboside monophosphate - ZRMP zeatin riboside monophosphate     

Changes in cytokinin activity during flower development in Cosmos sulphureus Cav

Endogenous cytokinin activity was determined in the flowers of Cosmos sulphureus Cav. from bud emergence to full bloom using the soybean callus bioassay. Cytokinin activity was low early in flower development but increased prior to full bloom. In Sephadex LH-20 column chromatography of flower extracts, the cytokinins present co-eluted with zeatin, zeatin riboside and glucoside cytokinin. While the former two predominated prior to full bloom, cytokinin glucoside activity appeared to be at a maximum at full bloom. The possible relevance of these findings is discussed in relation to flower development.     

Cytokinins of dryZea mays seed: Quantification by radioimmunoassay and gas chromatography-mass spectrometry

The endogenous cytokinins present in dryZea mays seed were determined using both radioimmunoassay and gas chromatography—mass spectrometry. Similar values for bases and ribosides were obtained by the two methods. The cytokinins present in embryo and endosperm were estimated separately using radioimmunoassay; similar levels of cytokinins were found in these two tissues. The major cytokinins detected on a whole-seed basis were dihydrozeatin riboside, O-glucosyldihydrozeatin riboside, zeatin 9-glucoside, zeatin, and the nucleotides of zeatin, dihydrozeatin, and isopentenyladenine. Cytokinin levels in the mature dry seed were considerably lower than cytokinin levels published in the literature for immature seed. Unexpected activity in the radioimmunoassays was detected in the wash from the DEAE cellulose column chromatography step. The compound(s) responsible for this activity did not have the solvent partitioning characteristics of a cytokinin base or riboside. They eluted as a single fraction following high-performance liquid chromatography on a Zorbax C8 column; this fraction showed no activity in theAmaranthus bioassay for cytokinins, but inhibited the activity of authentic zeatin riboside present at an optimal concentration.     

The effect of stratification on the endogenous levels of gibberellins and cytokinins in seeds of douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and sugar pine (Pinus lambertiana Dougl.)

Abstract Seeds of Douglas-fir and sugar pine which had been exposed to various periods of moist chilling (stratification) were extracted and bioassayed for gibberellin and cytokinin activity. In Douglas-fir a peak of gibberellin activity increased progressively during stratification and was seven times higher after 7 weeks, but declined subsequently. No change in the level of cytokinin activity was observed during stratification. However, a slight increase (1.7-fold) occurred in stratified seeds placed at 22°C for 3 days. In sugar pine a high level of gibberellin activity, approximately 20-fold higher than in unstratified controls, was present in extracts of seeds which had been stratified for 8 weeks. After 10 weeks of stratification cytokinin levels had increased approximately 200-fold. Four cytokinin-like substances were detected in seeds. Three of the substances are similar in their chromatographic properties to zeatin glucoside, zeatin riboside and zeatin, respectively. Both gibberellin and cytokinin activity declined rapidly after reaching maximum levels. In seeds with the coats removed, germination was rapid and complete in the absence of stratification. Prior to radicle emergence in coatless seeds cytokinin activity increased to a maximum (approximately a 1-7-fold increase) within 24 h. Significant levels of gibberellin-like substances were present after 8 h. Gibberellin activity could not be detected in coatless seeds which were imbibed in AMO-1618, and germination was inhibited significantly.     

Involvement of carbohydrates and cytokinins in pathogenicity ofHelminthosporium carbonum

Significant stimulation of the number of appressoria, penetration and colonization by conidia ofHelminthosporium carbonum occurred on decolorized maize leaves when exogenous carbohydrates and leaf leachates were added. Germination and germ tube length, however, did not exhibit appreciable differences on decolorized or non-decolorized maize leaves. Lower germination was recorded by leached conidia on decolorized leaves; while appressoria, penetration and colonization were absent. Addition of exogenous nutrients (sucrose>leaf leachates>yeast extract>glucose) enabled conidia to accomplish appressoria, penetration and colonization. Optimum levels for various nutrients observed were 2% (w/v) sucrose/glucose or 0.1% (w/v) yeast extract. Higher concentrations inhibited the infection stages of the pathogen. Depletion of host carbohydrates from green islands/infection sites adversely affect appressoria formation, penetration and colonization; and the loss of carbohydrates from the spore affects germination. Cytokinin-like activity at the infection site/green islands increased with the period of incubation of the host as compared to the surrounding tissue or tissue under water drops. The culture filtrate extracts ofH. carbonum recorded cytokinin-like activity which increased with growth of the fungus. TLC (thin layer chromatography) of cytokinin-like substances (tissue extract and culture filtrate) revealed major activity was confined to Rf zones 0.6 to 0.8 which co-chromatographed with zeatin and zeatin riboside. These substances increase at infection sites by virtue of which carbohydrates accumulate at these sites ensuring a continuous supply to the growing pathogen.     

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

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

Influence of Different Cytokinins on the Germination of Lettuce (Lactuca sativa) and Celery (Apium graveolens) Seeds

The naturally occurring cytokinins, zeatin, zeatin riboside and dihydrozeatin did not promote the germination of celery (Apium graveolens L.) seeds and 6-Δ²-isopentenyladenine (2iPA) and its riboside were only moderately active. Of the synthetic cytokinins, kinetin, kinetin riboside, and the disubstituted urea, N-phenyl-N′-pyridyl urea (NC5392) were moderately active, and 6-benzyl-aminopurine (BA) and its derivatives BA riboside and 6-benzyl-amino-9(tetrahydropyran-2yl)purine (SD8339) were the most active cytokinins tested. 6-(o-hydroxybenzyl)aminopurine (hyd-BA) and its naturally occurring riboside inhibited germination under normally inductive conditions. All the cytokinins examined were more active in promoting germination of lettuce (Lactuca sativa L.) than celery seeds. BA, BA riboside and SD8339 were again the most active cytokinins. In contrast to the results with celery, zeatin and zeatin riboside were highly active. The other cytokinins also showed high activity with the exception of dihydrozeatin, hyd-BA and hyd-BA riboside which were less active. Cytokinin ribosides were less active than the corresponding free bases during the early period of the lettuce seed incubation but total germination after 90 h was similar.     

甘蔗幼叶脱分化及愈伤组织形成过程中内源细胞分裂素和ABA的变化

甘蔗幼叶片,叶鞘和幼茎在含有2.4-D的培养条件下脱分化的情况有明显差异。它们除了在形态学上有区别外,这三种材料原有的细胞分裂素和ABA的水平明显不同。在幼叶片脱分化及愈伤组织形成过程中,内源细胞分裂素和ABA的水平也发生了明显变化。据此我们认为在甘蔗组织培养中2.4-D可能通过调节内源激素的水平及其相互作用,引起培养物中某些生理生化过程发生改变,从而进行脱分化和愈伤组织形成。