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

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

3 beta,13-dihydroxylated C20 gibberellins from inflorescences of Rumex acetosa L

Using full scan GC-MS a wide range of gibberellins (GAs) was identified in the young inflorescences of the dioecious species Rumex acetosa L., consistent with the ubiquitous early 13-hydroxylation pathway in both male and female plants. In addition, R. acetosa is the first species in which all three 3beta,13-dihydroxylated C(20)-GAs-GA(18), GA(38) and GA(23)-have been identified in the same organism, suggesting an early 3beta,13-dihydroxylation biosynthesis pathway in this species. Authentic GA(18), GA(38) and GA(23) were synthesized and their effects and that of GA(1), a GA common to both pathways, on the time to inflorescence emergence was investigated. GA(1) accelerated the emergence of inflorescences in both male and female plants. In addition some evidence for biological activity per se of the C(20)-GA(38) was obtained.     

Gibberellin Signal Transduction in Rice

Rumex acetosa L. (common sorrel) is a dioecious perennial in the family Polygonaceae. Gibberellins (GAs) of the early 13-hydroxylation pathway and the putative early 3, 13-hydroxylation pathway were previously identified in young R. acetosa inflorescences by GC-MS. In this investigation to examine the GA content of individual inflorescences ELISAs were used for quantitative analysis. Significant differences were revealed between the sexes in the GA content of young inflorescences, and GC-SRM was used to validate the observed trends. Males had higher levels of the 3, 13-hydroxylated C₂₀-GA GA₁₈ and the 2, 13-hydroxylated C₁₉-GA GA₂₉, whereas females had higher levels of the 13-hydroxylated C₂₀-GAs GA₅₃ and GA₁₉. It is suggested that the conversion from C₂₀-GAs to C₁₉-GAs is under tighter control in the inflorescences of females compared to male plants and therefore there is accumulation of the C₂₀-GAs in the females. Results from flowering bioassays using authentic GAs indicate that differences in GA content between the sexes are unlikely to be a consequence of sex determination.     

Developing Inflorescences of Male and Female <Emphasis Type="Italic">Rumex acetosa</Emphasis> L. Show Differences in Gibberellin Content

Rumex acetosa L. (common sorrel) is a dioecious perennial in the family Polygonaceae. Gibberellins (GAs) of the early 13-hydroxylation pathway and the putative early 3, 13-hydroxylation pathway were previously identified in young R. acetosa inflorescences by GC-MS. In this investigation to examine the GA content of individual inflorescences ELISAs were used for quantitative analysis. Significant differences were revealed between the sexes in the GA content of young inflorescences, and GC-SRM was used to validate the observed trends. Males had higher levels of the 3, 13-hydroxylated C₂₀-GA GA₁₈ and the 2, 13-hydroxylated C₁₉-GA GA₂₉, whereas females had higher levels of the 13-hydroxylated C₂₀-GAs GA₅₃ and GA₁₉. It is suggested that the conversion from C₂₀-GAs to C₁₉-GAs is under tighter control in the inflorescences of females compared to male plants and therefore there is accumulation of the C₂₀-GAs in the females. Results from flowering bioassays using authentic GAs indicate that differences in GA content between the sexes are unlikely to be a consequence of sex determination.     

Changes in Gene Expression from Diploid to Autotetraploid Status of Lycopersicon esculentum

The cytokinin activity of the root exudate, the leaves, and the apices of vegetative and flowering white lupin plants (Lupinus albus L.) was investigated. The level of cytokinin activity in the root exudate decreased over the 11-week experimental period. Four peaks of cytokinin activity were recorded in the root exudate of 8-week-old plants after fractionation on Sephadex LH-20. Two of these peaks co-eluted with zeatin and zeatin riboside. It is suggested that the remaining peaks represent nucleotide and glucoside cytokinins. The cytokinin levels in extracts of the mature leaves fluctuated slightly over the experimental period. Three peaks of activity co-eluting with zeatin, zeatin riboside and the glucoside cytokinins were recorded in extracts of mature leaves of 8-week-old plants. In the apices cytokinin activity could only be detected in the inflorescences of flowering plants. It would appear that cytokinins produced by the roots accumulate in the fully expanded mature leaves, but are utilized in the rapidly growing apical region and in young expanding leaves.     

Cytokinins in the vascular saps of Ricinus communis

Roots are recognised as the major sites of cytokinin synthesis and shoots receive a continuous supply of cytokinins from the roots. Although reports are available on the xylem mobility of putative free bases and their ribosides, relatively few studies on the phloem mobility of cytokinins have been reported. The origin of phloem-mobile cytokinins is uncertain but there is evidence which implicates a recirculation from the root source. This study is the first report in which zeatin and zeatin riboside from the root pressure exudate and phloem sap of Ricinus have been identified by full-scan GC-MS and quantified by GC-MS selective-ion-monitoring. In this study, the concentration of cytokinins in root pressure exudate was similar, but lower, and in the phloem sap higher than that reported previously. The concentration of cytokinins quantified in the phloem sap confirms their transport in the sieve tubes. The relatively high concentration of zeatin riboside detected in the root pressure exudate and of zeatin detected in the phloem sap indicate a possible vascular recirculation of these hormones.     

Cytokinin Differences in In Vitro Cultures and Inflorescences from Normal and Mantled Oil Palm (Elaeis guineensis Jacq.)

The mantled abnormality phenotype of the oil palm affects fruit development and thus jeopardizes oil yield. Cytokinins have been implicated in the development of the mantled phenotype. Endogenous cytokinin levels in the normal and mantled phenotypes were compared to determine whether levels of specific cytokinins are associated with mantling. Endogenous cytokinins were identified and quantified in in vitro cultures and inflorescences from normal and mantled oil palms. Twenty-two isoprenoid cytokinins, comprising the zeatin, dihydrozeatin, and isopentenyladenine types, were quantified. Total cytokinin levels, particularly of trans-zeatin and isopentenyladenine types, increased during the in vitro culture process, with the highest levels detected at the proliferating polyembryoid stages. The cytokinins were present mainly in their inactive 9-glucoside forms during in vitro culture. On the other hand, the predominant trans-zeatin cytokinins in inflorescences were present mainly in their ribotide forms, suggesting a metabolic pool of cytokinins for conversion to biologically active free bases or ribosides. Levels of specific cytokinins were significantly different in tissues at different stages. Mantled developed inflorescences contained higher levels of isopentenyladenine 9-glucoside compared with normal inflorescences. Mantled-derived callus tissues had higher isopentenyladenine levels but significantly lower levels of trans-zeatin 9-glucoside, dihydrozeatin riboside, and dihydrozeatin riboside 5′-monophosphate cytokinins compared with normal-derived callus. It would be of considerable interest to verify these specific cytokinin differences in more callus cultures and clones.     

Correlation of Endogenous Gibberellic Acid with Initiation of Mango Shoot Growth

Stems of mango (Mangifera indica L.) rest in a nongrowing, dormant state for much of the year. Ephemeral flushes of vegetative or reproductive shoot growth are periodically evoked in apical or lateral buds of these resting stems. The initiation of shoot growth is postulated to be primarily regulated by a critical ratio of root-produced cytokinins, which accumulate in buds and by leaf-produced auxin, which decreases in synthesis and transport over time. Exogenously applied gibberellic acid (GA3) delays initiation of bud break but does not determine whether the resulting flush of growth is vegetative or reproductive. We tested the hypothesis that endogenous GA3, which influences release of these resting buds, may decrease in stem tips or leaves with increasing age of mango stems. GA3 and several other GAs in stem tip buds and leaves were identified and quantified in stems of different ages. The major endogenous GAs found in apical buds and leaves of vegetative mango stems were early 13-hydroxylation pathway gibberellins: GA1, epi-GA1, GA3, GA19, GA20, and GA29, as identified by gas chromatography-mass spectrometry (GC-MS). A novel but unidentified GA-like compound was also present. The most abundant GAs in apical stem buds were GA3 and GA19. Contrary to the hypothesis, the concentration of GA3 increased within buds with increasing age of the stems. The concentrations of other GAs in buds were variable. The concentration of GA3 did not change significantly with age in leaves, whereas that of most of the other GAs declined. GA1 levels were greatest in leaves of elongating shoots. These results are consistent with the concept that rapid shoot growth is associated with synthesis of GAs leading to GA1. The role of GA3 in delaying bud break in mango is not known, but it is proposed that it may enhance or maintain the synthesis or activity of endogenous auxin. It, thereby, maintains a high auxin/cytokinin ratio similar to responses to GA3 that maintain apical dominance in other plant species.     

Dynamics of growth and the content of endogenous phytohormones during kidney bean scoto-and photomorphogenesis

The dynamics of growth and the contents of free and bound endogenous IAA, gibberellins (GA), cytokinins (zeatin and its riboside), and ABA in kidney bean plants (Phaseolus vulgaris L., cv. Belozernaya) grown in darkness or in the light was studied. Phytohormones were quantified in 5–15-day-old plants by the ELISA technique. Plant growth and phytohormone content were shown to depend on plant age and the conditions of illumination. During scotomorphogenesis, changes in the biomass and hypocotyl length were highly correlated with the content of GA, whereas during photomorphogeneses, these parameters were correlated with the content of zeatin. In darkness, epicotyl growth displayed a positive correlation with the content of GA, whereas in the light, the correlation was negative. Growth characteristics of the primary leaves were shown to correlate with IAA in darkness and with GA and zeatin in the light. At a low concentration of cytokinins in illuminated leaves, cell divisions occurred, whereas, at the higher cytokinin concentrations, cell expansion occurred. The highest content of GA was characteristic of leaves in the period of growth cessation. ABA accumulated during active leaf and root elongation and biomass increment in the light and during hypocotyl growth in darkness. After plant illumination, the ratio of auxins to cytokinins increased in bean roots and decreased in their epicotyls. Thus, light changed the developmental programs of bean plants, which was manifested in the changed rate and duration of growth of various organs (root, hypocotyl, epicotyl, and leaf). Some mechanisms of light action depended on the contents of IAA, ABA, GA, and cytokinins and the ratios between these phytohormones. Differences between scotonorphogenesis of mono-and dicotyledonous plants are discussed in relation to the levels of phytohormones in them.     

Correlation of Endogenous Gibberellic Acid with Initiation of Mango Shoot Growth

Stems of mango (Mangifera indica L.) rest in a nongrowing, dormant state for much of the year. Ephemeral flushes of vegetative or reproductive shoot growth are periodically evoked in apical or lateral buds of these resting stems. The initiation of shoot growth is postulated to be primarily regulated by a critical ratio of root-produced cytokinins, which accumulate in buds and by leaf-produced auxin, which decreases in synthesis and transport over time. Exogenously applied gibberellic acid (GA3) delays initiation of bud break but does not determine whether the resulting flush of growth is vegetative or reproductive. We tested the hypothesis that endogenous GA3, which influences release of these resting buds, may decrease in stem tips or leaves with increasing age of mango stems. GA3 and several other GAs in stem tip buds and leaves were identified and quantified in stems of different ages. The major endogenous GAs found in apical buds and leaves of vegetative mango stems were early 13-hydroxylation pathway gibberellins: GA1, epi-GA1, GA3, GA19, GA20, and GA29, as identified by gas chromatography-mass spectrometry (GC-MS). A novel but unidentified GA-like compound was also present. The most abundant GAs in apical stem buds were GA3 and GA19. Contrary to the hypothesis, the concentration of GA3 increased within buds with increasing age of the stems. The concentrations of other GAs in buds were variable. The concentration of GA3 did not change significantly with age in leaves, whereas that of most of the other GAs declined. GA1 levels were greatest in leaves of elongating shoots. These results are consistent with the concept that rapid shoot growth is associated with synthesis of GAs leading to GA1. The role of GA3 in delaying bud break in mango is not known, but it is proposed that it may enhance or maintain the synthesis or activity of endogenous auxin. It, thereby, maintains a high auxin/cytokinin ratio similar to responses to GA3 that maintain apical dominance in other plant species.     

Comparison of endogenous cytokinins and cytokinin oxidase/dehydrogenase activity in germinating and thermoinhibited Tagetes minuta achenes

Tagetes minuta L. achenes are thermoinhibited at temperatures above 35°C and have accelerated radicle emergence (germination) when subsequently transferred to an optimal temperature (25°C). Endogenous cytokinins and cytokinin oxidase/dehydrogenase (CKX) activity were compared in normally germinating (25°C) and thermoinhibited (72h at 36°C then transferred to 25°C) T. minuta achenes. Following imbibition, endogenous cytokinin concentrations changed in normally germinating T. minuta achenes, with a gradual decrease in dihydrozeatin-type (DHZ) cytokinins, a large increase in cis-zeatin-type (cZ) cytokinins, a smaller increase in N?-(2-isopentenyl)adenine-type (iP) cytokinins and a peak of trans-zeatin-type (tZ) cytokinins at 13 h. These changes in the isoprenoid cytokinin profile were similar in the thermoinhibited achenes imbibed at 36°C, despite the thermal block preventing radicle emergence. The exception was the iP-type cytokinins that only increased when transferred to 25°C. Profiles of the physiologically active free bases showed an increase in tZ prior to radical emergence in both normally germinating (13 h) and thermoinhibited achenes. A large transient peak in aromatic cytokinins [N?-benzyladenine-type (BA)] occurred during early seedling establishment in normally germinating achenes (40 h) while a transient maximum in BA-type cytokinins was found prior to radicle emergence in the thermoinhibited achenes (24 h). The CKX activity was enhanced in normally germinating achenes as the cytokinin concentration increased following imbibition. In thermoinhibited achenes, an elevated temperature negatively affected the CKX activity that only increased when the achenes were transferred to 25°C, corresponding to an increase in iP-type cytokinins. However, the favored cytokinin deactivation pathway in T. minuta appears to be 9-glycosylation, as 9-glucosides accounted for over 50% of the total cytokinin pool in both normal and thermoinhibited achenes.     

Effects of Temperature and Growth Regulators on Formation of Anlagen, Tendrils and Inflorescences in Vitis vinifera L.

The effects of single, combined and sequential application ofgibberellic acid (GA), chlormequat and cytokinins on the formationof anlagen, tendrils and inflorescences were studied in grapevines(cv. Muscat of Alexandria) grown with natural illumination athigh temperature (30 °C day to 25 °C night) and at lowtemperature (21 °C day to 16°C night or 18 °C dayto 13 °C night). GA promoted the formation of anlagen andgrowth of tendrils regardless of temperature, but inhibitedinflorescence production. Chlormequat had the opposite effecton anlagen formation and tendril growth and promoted inflorescenceformation from pre-formed anlagen or from tendril initials.While low temperature is normally unfavourable for inflorescenceformation, this was induced by chlormequat even at low temperature,but only with summer light conditions. Cytokinin application to plants pre-treated with chiormequatcaused tendrils to grow into inflorescences regardless of temperatureregimes. Moreover, shoot primordia were also formed in place⁸oftendrils in cytokinin treated plants even without chlormequatpre-treatment. Vitis vinifera L., grapevine, gibberellic acid, chlormequat, cytokinins, benzyladenine, inflorescence, tendrils     

Hormonal Characterization of Transgenic Tobacco Plants Expressing the rolC Gene of Agrobacterium rhizogenes TL-DNA

Transgenic tobacco (Nicotiana tabacum L. cv Wisconsin 38) plants expressing the Agrobacterium rhizogenes rolC gene under the control of the cauliflower mosaic virus 35S RNA promoter were constructed. These plants displayed several morphological alterations reminiscent of changes in indole-3-acetic acid (IAA), cytokinin, and gibberellin (GA) content. However, investigations showed that neither the IAA pool size nor its rate of turnover were altered significantly in the rolC plants. The biggest difference between rolC and wild-type plants was in the concentrations of the cytokinin, isopentenyladenosine (iPA) and the gibberellin GA19. Radio-immunoassay and liquid chromatography-mass spectrometry measurements revealed a drastic reduction in rolC plants of iPA as well as in several other cytokinins tested, suggesting a possible reduction in the synthesis rate of cytokinins. Furthermore, gas chromatography-mass spectrometry quantifications of GA19 showed a 5- to 6-fold increase in rolC plants compared with wild-type plants, indicating a reduced activity of the GA19 oxidase, a proposed regulatory step in the gibberellin biosynthesis. Thus, we conclude that RolC activity in transgenic plants leads to major alterations in the metabolism of cytokinins and gibberellins.     

Gibberellin-induced delay of leaf senescence of Alstroemeria cut flowering stems is not caused by an increase in the endogenous cytokinin content

The effects of various chemically pure gibberellins and cytokinins on leaf yellowing of Alstroemeria were described. The loss of chlorophyll was measured both in leaves of cut flowering stems and in a model system consisting of detached leaf tips. It was demonstrated that plant growth substances affected chlorophyll loss in both systems to the same extent. Leaf senescence was delayed by various gibberellins and cytokinins. The results demonstrated that some of the gibberellins (GA₄ and GA₇) are far more effective in delaying chlorophyll loss than GA₃, which is commonly used as a postharvest treatment for Alstroemeria cut flowering stems. Immunoassays were used to demonstrate that the effect of gibberellins on leaf yellowing does not involve an increase in the endogenous cytokinin concentrations in the leaves as an intermediate step.Abbreviations GA gibberellin A - HPLC high performance liquid chromatography - GA_3Mc GA₃-methyl ester - ZR zeatin riboside - IPAR isopentenyl adenine riboside.     

Cytokinins in macroalgae

Thirty-one seaweeds were collected from the warmer KwaZulu-Natal coast and the cooler Cape waters (South Africa). Plant material was extracted with 70% ethanol supplemented with deuterium labelled standards of all known isoprenoid cytokinins. The samples were then centrifuged and purified by combined DEAE-Sephadex×octadecylsilica column and immunoaffinity chromatography and finally analysed for cytokinins by HPLC-linked mass spectrometry and a photodiode array detector. The cytokinin profiles were similar in all the macroalgae regardless of their taxonomy and growing locality. The main type of isoprenoid cytokinins present were zeatins with cis forms being more common than trans forms and isopentenyladenine (iP) derivatives. Only a few dihydrozeatin-type cytokinins were detected at very low levels in only nine species. Aromatic cytokinins were also present but at lower levels and were represented by benzyladenine (BA) and ortho- and meta-topolin derivatives. The topolins were present in greater diversity and concentrations than BA. For all the cytokinin types, the free bases, O-glucosides and nucleotides were the most common with no N-glucosides being detected and ribosides present at very low levels. The results suggest that different pathways for regulating cytokinin concentrations operate in macroalgae than in higher plants.     

Cytokinins in Rosa hybrida in relation to bud break

To assess the role of endogenous cytokinins in growth and development of Rosa hybrida , their concentrations in bleeding sap and in roots, stem, leaves, axillary shoots and bottom breaks in three stages of development were quantified. Cytokinins were purified by means of immunoaffinity chromatography and HPLC, and identified by retention time, UV spectrum and GC-MS. The major translocation form in the xylem was zeatin riboside (ZR). In all mature tissues, cytokinins of the zeatin-type were predominant, amounting to 80–90% of the total cytokinin concentration. The stems contained high concentrations of cytokinins, probably caused by lateral movement of ZR from the xylem to adjacent stem tissue and the ability of the stem to metabolize cytokinins. In young leaves the contribution of isopentenyl adenine (iP)-type cytokinins to the total cytokinin pool was about 50%, indicating that these leaves might be capable of de novo synthesis of cytokinins. In older leaves, the concentration of an unidentified cytokinin-like compound increased to more than 50% of total cytokinins. This compound, which was also found in the roots, might be a storage form of cytokinins. In young axillary shoots, about 50% of the cytokinins are iP-compounds, suggesting either import of iP-type cytokinins via the phloem or de novo synthesis of cytokinins. In buds forming bottom breaks, ZR and zeatin riboside monophosphate (ZRMP) are the main cytokinins, indicating that these buds receive their cytokinins from the roots.