Cytokinins in photoperiodic induction of flowering in Chenopodium species

Changes in cytokinin (zeatin – Z, zeatin riboside – ZR, isopentenyladenine – iP, isopentenyladenosine – iPA) levels were determined under light regimes inductive and non-inductive for flowering in leaves, stems, roots and apical parts of short-day Chenopodium rubrum and long-day Chenopodium murale. In leaves. stems and roots of both plant species the level of cytokinins (in C. rubrum of Z and ZR, in C. murale of Z. ZR, iP and iPA) decreased by about 50% during the dark period and increased again during the subsequent light period, No significant changes in cytokinin levels were observed in continuous light. In apical parts of C. rubrum cytokinin level (Z, ZR, iP) was dramatically increased (by 400–500%) at the end of the dark period and decreased to about the original value during the following light period, while no changes were observed in continuous light. In apical parts of C. murale the level of cytokinins doubled during floral induction consisting of 10 days of continuous light. A red (R) break (15 min at the 6th h of darkness), which prevents flowering in C. rubrum , has no significant effect on cytokinin levels in leaves at the end of darkness. Cytokinin levels increased 1 h after R and decreased again rapidly. On the other hand, the increase of cytokinin level in the apical parts of C. rubrum was largely prevented by the R break. These effects of R on cytokinin levels were not reverted by far-red (FR), while the effect on flowering was reverted. It may be concluded that there is no correlation between changes in cytokinin levels in leaves. Stems and roots and photoperiodic flower induction, as both species, representing different photoperiodic types, showed similar changes under the same light regime. The increase of cytokinin levels in apical parts of both photoperiodic species during floral induction suggests a role (increased cell division and branching) for cytokinins in apex evocation.     

Distribution of cytokinin-like activity in different plant parts of the water hyacinth, Eichhornia crassipes

Cytokinin-like activity in extracts of leaf laminae, petioles, shoots, roots and flowers of young plants of the water hyacinth, Eichhornia crassipes S. was analyzed following Sephadex LH-20 column chromatography using the soybean callus bioassay. In all plant parts analyzed, two prominent peaks of cytokinin activity having elution volumes similar to zeatin and zeatin riboside were detected. Putative cytokinin gluco-side-like activity was detected only in leaves and flowers. The cytokinin complements of the leaves and the roots were qualitatively different. It would appear that cytokinins supplied by the roots are metabolized in the leaves or certain cytokinins are synthesized in the leaves themselves. The possible significance and distribution of cytokinins in different plant parts in relation to roots is discussed.     

Cytokinins in Xanthium strumarium L.: Distribution in the Plant and Production in the Root System

Cytokinins from leaf laminae, buds, petioles, stems, roots,and root exudate of mature vegetative plants of Xanthium strumariumL. were extracted, fractionated, and partially characterizedby means of column chromatography with Sephadex LH20. Two peaksof cytokinin activity with elution volumes corresponding tozeatin and zeatin riboside were detected, in varying concentrations,in all plant parts. A third cytokinin, detected only in petiolesand in expanding and mature leaves, eluted off the Sephadexcolumn before zeatin riboside. This cytokinin (peak ‘a’)was converted to zeatin or to a zeatin-like cytokinin followingboth acid hydrolysis and treatment with ß-glucosidase.Peak ‘a’ was not detected in buds or in the youngestdeveloping leaves but was the predominant cytokinin presentin half-expanded and more mature leaves. By contrast, the zeatinriboside-like peak (peak ‘b’) constituted the majorcytokinin in root exudate, apical buds, and the youngest developingleaves, while not greatly contributing to the cytokinin contentof mature leaves. The detopped root system was shown to be capable of cytokininproduction. The distribution of cyrtokinins in the plant isdiscussed in relation to their probable origin in the root system.     

Auxins and cytokinins exuded during formation of roots by detached primary leaves and stems of dwarf French bean (Phaseolus vulgaris L.)

Summary Hypocotyls of detached stems standing in culture solution produced adventitious roots sooner than did petioles of detached primary leaves. An auxin, probably indol-3-ylacetic acid, appeared in the solutions before the hypocotyls or petioles produced roots. After attaining a maximum, the amounts of auxin in the solutions decreased as fewer roots were formed. Two cytokinins were found in the culture solutions; one had a similar R_f to zeatin, the other ran more slowly on chromatograms. The amounts of cytokinin in the solutions were associated with root formation. Stems soon died unless their hypocotyls formed roots, but the primary leaves survived without roots forming provided a callus formed on the petiole. Hence adventitious roots, or callus tissues, may have produced cytokinins that replaced those produced by the original roots, found in sap exuded from the stem stumps, and were essential for survival of the stems and leaves.     

APOLAR MOVEMENT OF ZEATIN THROUGH COLEUS PETIOLES AND PISUM ROOTS AS ESTIMATED BY BIOASSAY AND RADIOACTIVE LABELLING

The movement and polarity of zeatin, a highly active, endogenous cytokinin, through petioles and roots were tested in the classical experimental arrangement using excised 5-mm sections. Zeatin in the receiver cylinders of agar was measured by soybean callus bioassay and by liquid scintillation counting of ¹⁴C that had been added in the donor cylinders as [8-¹⁴C] zeatin. Both methods agreed in showing movement, but there was no polarity in Coleus #5 petioles. The amounts moved were about one-tenth of the GA-3 movement through petioles of the third pair of leaves of the same clone. Movement of ¹⁴C-zeatin through Pisum roots was similarly statistically significant but non-polar; the amounts moved were similar to those previously observed for polar GA-3 movement through Zea roots.     

Changes in the Content of Indole-3-Acetic Acid and Cytokinins in Spruce, Fir and Oak Trees after Herbicide Treatment

Treatment of spruce, fir and oak trees with herbicides, which may be one of the forest damage inducing agents, caused pronounced changes in the contents and distribution of indole-3-acetic acid (IAA) and cytokinins (CKs) one year after treatment, i.e. at the time of the first microscopically visible damage in treated trees. In Picea pungens IAA content increased in the terminal buds by about 105 % and in the apical buds of the first order branches by 220 %. The same was true for young sprouts of Abies nordmanniana, while in leaves of oak trees IAA content was decreased by 15 % after glyphosate treatment and by 30 % after 2,4-dichlorophenoxyacetic acid (2,4-D) treatment. Another striking feature was a significantly decreased content of IAA in the lower parts of roots in Picea pungens (50 % of the control), which is accompanied by an increase in IAA content in the middle part of the roots (130 %). On the other hand, the IAA content of both sprouts and roots of A. nordmanniana was significantly increased after herbicide treatment.In P. pungens, the content of free cytokinins (sum of zeatin, zeatin riboside, isopentenyladenine and isopentenyladenosine) decreased due to herbicide treatment. The strongest decrease was seen in roots, especially in their upper and middle parts (the average reduction of cytokinin content in roots was 63 %). In the above-ground organs the reduction was seen namely for isopentenyladenine and isopentenyladenosine, while the abundance of zeatin riboside was, on the other hand, higher in treated plants. In Quercus robur leaves, the total content of cytokinins also decreased, namely after glyphosate treatment. In consequence of these changes, CK/IAA ratio decreased pronouncedly in all organs of herbicide-treated trees, with the exception of oak leaves treated by 2,4-D.     

Hybrid Weakness in Phaseolus vulgaris L. I. Disruption of Development and Hormonal Allocation

A reduced concentration of cytokinins may cause the abnormal growth and development found in F1 hybrids between Andean and Mesoamerican races of Phaseolus vulgaris L. In this study, concentrations of the transportable cytokinin zeatin riboside (ZR) were measured by ELISA for ZR (cross reactivities dihydrozeatin, 14%, zeatin 7.6%) in roots, stems, and leaves of a Phaseolus Mesoamerican landrace (P. vulgaris L. cv. Redkloud), an Andean landrace (P. vulgaris L. cv. Batt), and their F1 hybrids. Concentrations of ZR in roots and leaves of F1 hybrids were significantly less than that found in roots and leaves of parental cultivars. Approximately 90% of the ZR found in F1 hybrids was found sequestered in the stems, whereas cytokinins of the parental cultivars were distributed throughout the plant (roots: Batt 37%, Redkloud, 44%; stems: Batt 35%, Redkloud 42%; leaves: Batt 28%, Redkloud 14%). These results suggest that abnormal growth and development of F1 hybrids may involve interruption of the regulation of cytokinin allocation, thereby disrupting the root-shoot feedback loop between root-sourced cytokinins and putative shoot-produced factors. Received October 15, 1998; accepted May 12, 1999     

Morphogenesis of potato plants in vitro. II. Endogenous levels,distribution, and metabolism of IAA and cytokinins

The levels of endogenous IAA and cytokinins (zeatin, zeatin riboside, isopentenyladenine, and isopentenyladenosine) were determined in potato plants cultured in vitro under red light (R) and blue light (B) on medium with or without hormones. On medium without hormones in B, plants contained much higher cytokinin levels, particularly in leaves and roots, and also slightly elevated IAA levels. Kinetin in the medium in B changed the distribution of cytokinins and significantly increased IAA level in roots. In R, the presence of kinetin led to an increased cytokinin level in the whole plant, while the IAA level was slightly lower. IAA in the medium in B decreased cytokinin level in all plant parts, while the IAA level did not change significantly. In R, the presence of IAA in the medium led to a moderate increase of CK level and to a significant increase in IAA level, especially in roots. Uptake of 1-¹⁴C-IAA and of ³H-zeatin was generally higher in B than in R. Higher percentage of IAA taken up in B was converted to conjugates in the roots. Metabolism of ³H-zeatin was similar in R and B with only slight differences in metabolite amounts.Thus, in all experimental situations in which tuber formation was stimulated, IAA level in roots and stolons rose significantly, stressing the importance of an IAA gradient for tuber formation.     

Mass spectrometric analysis of cytokinins in plant tissues : v. Identification of the cytokinin complex of datura innoxia crown gall tissue

The cytokinin complex of Datura innoxia Mill. crown gall tissue was purified by ion exchange, Sephadex LH-20 chromatography and reversed-phase high performance liquid chromatography. By gas chromatography-mass spectrometry using ²H-labeled compounds, the following cytokinins were identified in the basic fraction eluting from a cation exchange column: zeatin, zeatin riboside, dihydrozeatin, dihydrozeatin riboside, their corresponding O-glucosides, 7- and 9-glucosides of zeatin, 9-glucoside of dihydrozeatin, isopentenyladenine, and isopentenyladenosine. Zeatin riboside 5′-monophosphate was the major cytokinin nucleotide in the tissue. In addition, dihydrozeatin riboside and isopentenyladenosine were identified in the nucleotide fraction following enymic degradation.     

Seasonal Changes in the Cytokinin Content of the Leaves of Salix babylonica

The young and old leaves of Salix babylonica contain at least four cell division-inducing compounds which coeluted with zeatin, zeatin riboside and their glucosylated derivatives. During the course of the growing season quantitative changes in the cytokinin content of the leaves were observed. The cytokinin glucosides increased as the leaves aged. The compounds which co-chromatographed with zeatin and zeatin riboside initially increased until early autumn and then decreased as the leaves senesced. It appears as though the cytokinins transported from the roots are metabolized in the leaves and are converted to their glucosides. Although it has been reported in the literature that Salix root exudate contains very small amounts of cytokinin in late summer and autumn, these compounds increase in the leaves for most of the growing season, suggesting that the leaves may not only obtain cytokinins from the roots but may well be an active site of cytokinin synthesis. It is, however, possible that cytokinins are also transported to the leaves via the phloem, thus accounting for their accumulation in these organs.     

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.     

Dynamics of Cytokinins in Apical Shoot Meristems of a Day-Neutral Tobacco during Floral Transition and Flower Formation

This study considered cytokinin distribution in tobacco (Nicotiana tabacum L.) shoot apices in distinct phases of development using immunocytochemistry and quantitative tandem mass spectrometry. In contrast to vegetative apices and flower buds, we detected no free cytokinin bases (zeatin, dihydrozeatin, or isopentenyladenine) in prefloral transition apices. We also observed a 3-fold decrease in the content of cytokinin ribosides (zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine) during this transition phase. The group concluded that organ formation (e.g. leaves and flowers) is characterized by enhanced cytokinin content, in contrast to the very low endogenous cytokinin levels found in prefloral transition apices, which showed no organogenesis. The immunocytochemical analyses revealed a differing intracellular localization of the cytokinin bases. Dihydrozeatin and isopentenyladenine were mainly cytoplasmic and perinuclear, whereas zeatin showed a clear-cut nuclear labeling. To our knowledge, this is the first time that this phenomenon has been reported. Cytokinins do not seem to act as positive effectors in the prefloral transition phase in tobacco shoot apices. Furthermore, the differences in distribution at the cellular level may be indicative of a specific physiological role of zeatin in nuclear processes.     

Changes in the Cytokinins of Radish Roots during Maturation

The cytokinins of developing radish roots were extracted, partially purified, and separated by thin-layer chromatography into three distinct bands of activity. One band was identified chromatographically as zeatin ribonucleotide and another was indistinguishable from a mixture of zeatin and zeatin ribonucleoside. The third band was not identified, but it was not a derivative of zeatin or of isopentenyladenine. The unidentified cytokinin had physiological properties quite different from those of the zeatin derivatives. The zeatin-based cytokinins increased in radish roots with the onset of cambial activity, and reapplication of these cytokinins to cultured primary roots stimulated cambial activity. The unidentified cytokinin became abundant only after extensive secondary thickening had occurred, and it was localized almost entirely in the xylem. It did not stimulate cambial activity in cultured roots. The evidence indicates that zeatin and its derivatives regulate cambial activity in radish, and that the unidentified cytokinin may be synthesized in the roots and transported to the shoot.     

Irradiance-induced alterations of growth and cytokinins in Phaseolus vulgaris seedlings

Light is a major environmental factor affecting plant growth and development. The cytokinins have many similar effects on these processes and may be involved in photomorphogenesis. In order to study the correlation between light and endogenous cytokinins, we have examined growth parameters and endogenous cytokinins in stems, leaves and other organs of Phaseolus vulgaris, cultivated for 10 days under a range of irradiances (25, 110, 350 and 500 µmol m^–2 s^–1). The nucleotides isopentenyladenosine-5-monophosphate and zeatin riboside-5-monophosphate were the dominant cytokinins, whereas both free bases and ribosides were below the detection level (0.5 pmol g^–1). Plants grown at the highest irradiance had in their stems, leaves, petioles and roots significantly higher levels of cytokinins than had plants grown at the lowest irradiance. As expected, increased light influx increased the dry weight of the root, petiole and leaf, and increased the leaf area, with concomitant increases in the cytokinins in these plant parts. However, the stem showed a different and more complex relationship with irradiance. Stem cytokinin levels increased drastically between 350 and 500 µmol m^–2 s^–1, but this was not correlated with any change in stem length; the light inhibition of stem elongation was mainly seen when irradiance was increased to 110 µmol m^–2 s^–1. Taken as a whole, the results are consistent with an effect of irradiance and cytokinins on the processes favouring biomass production.     

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.     

Influence of Different Cytokinins on the Transpiration and Senescence of Excised Oat Leaves

In order to investigate the possibility that cytokinins control transpiration indirectly through affecting leaf senescence, a direct comparison was made of the effect of different cytokinins on transpiration and senescence of oat leaves (Avena sativa L. cv. Forward). Senescence was assessed by measuring chlorophyll loss. The synthetic cytokinins N⁶ benzyladenine (BA) and kinetin delayed senescence and increased transpiration of oat leaves to a greater extent than did the naturally occurring compounds zeatin, N^b-Δ² isopentenyladenine (i⁶ Ade) and 6-ø-hydroxybenzyladenosine (hyd-BA riboside). During the early stages of the transpiration experiment zeatin showed similar or greater activity than BA. This period was longest when freshly excised leaves were used, was reduced when leaves were used after incubation in distilled water in the dark for 20 h and was eliminated by incubation in cytokinin solution in the dark. After this period the activity of zeatin declined relative to BA. The effect of cytokinins in increasing transpiration occurred only in the light; no effect was observed in the dark. BA showed higher activity than zeatin in senescence tests but both cytokinins were less effective as the tests progressed, this decrease in activity being more rapid when older leaves were used. The results are discussed in relation to the mechanisms by which endogenous cytokinins might control sensecence and transpiration in oat leaves and to the value of the oat leaf senscence and transpiration bioassays as tests for cytokinin activity of plant extracts.     

Dynamics of Endogenous Cytokinins during the Growth Cycle of a Hormone-Autotrophic Genetic Tumor Line of Tobacco

The profile of endogenous cytokinins in a genetic tumor line of tobacco, namely, Nicotiana glauca (Grah.) × Nicotiana langsdorffii (Weinm.), following 1 to 10 weeks of growth on solid medium was determined by radioimmunoassay. ³H-labeled cytokinins of high specific activity were added during tissue extraction to correct for the purification losses. Following subculture (of 4-week-old tissues when their cytokinin content is high) onto fresh medium the total cytokinin content continued to be high during the first week (1470 picomoles per gram fresh weight) when the tissue fresh weight remained essentially unchanged (lag phase). The cytokinin levels then declined by about half in 2- and 3-week-old tissues (626 and 675 picomoles per gram fresh weight, respectively), a period when rapid increase in tissue fresh weight was recorded. Increments of 840% and 2780% over initial fresh weight were obtained in 2- and 3-week-old cultures, respectively. The cytokinin content then increased to initial high levels in 4-week-old tissues (1384 picomoles per gram fresh weight) after which it gradually declined with tissue age. The lowest cytokinin levels (432 picomoles per gram fresh weight) were observed in 10-week-old tissues. Maximal tissue fresh weight (4030% increase over initial fresh weight) was recorded in 5-week-old cultures after which it decreased slowly to 77.5% of the highest tissue fresh weight in 10-week-old cultures. Zeatin appeared to be the dominant endogenous cytokinin in tissues of all ages. Other cytokinins quantified were dihydrozeatin, zeatin riboside, and dihydrozeatin riboside; the values may include contributions from aglucones derived from the hydrolysis of corresponding O-glucosides, since the entire basic fraction was treated with β-glucosidase before analysis. In addition the levels of isopentenyladenine, isopentenyladenosine, and the nucleotides of zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine were also determined.     

The Contents of Abscisic Acid and Cytokinins in Wild-Growing Species Using Different Ecological Strategies

The biomass structure and the contents of (ABA) and cytokinins (zeatin plus zeatin riboside) were studied in flowering plants of ten herbaceous species of the Middle Urals representing stress-tolerant (S) and ruderal (R) ecological strategies. The plants, which explicitly manifested S- and R-strategies, differed in ABA and cytokinin concentrations and phytohormone partitioning in particular organs. Higher ABA accumulation in the leaves and especially in the reproductive organs (here the ABA concentration exceeded by several times that of cytokinins) was characteristic of the R-plants as compared to the S-plants. In addition, the R-plants manifested higher cytokinin contents in the roots, whereas, in the S-plants, cytokinins dominated in the leaves and the reproductive organs (in the latter, the content of cytokinins exceeded that of ABA). The diverse hormonal status of the R- and S-species is discussed in relation to the patterns of plant growth and development and the photosynthate transport and partitioning characteristic of two ecological strategies. The authors conclude that plant ability to maintain the typical hormonal balance is instrumental in acquiring specific ecological strategies.     

Alteration of Hormonal Levels in a Rootless Epiphytic Bromeliad in Different Phenological Phases

Major changes in indole-3-acetic acid (IAA) and cytokinin (CK) levels occur at different phenological phases of Tillandsia recurvata shoots. This epiphytic rootless bromeliad was chosen as suitable material for hormonal analysis because CK synthesis is restricted to the shoots, thus avoiding problems in the interpretation of results caused by translocation and interconversion of CK forms between roots and leaves encountered in plants with both organs. Young plants of T. recurvata have weak apical dominance because side shoots appeared early in development, and branch growth was correlated with a strong increase in the level of zeatin. The flowering phase was characterized by a significant increase in free base CKs, zeatin, and isopentenyladenine compared with the levels found in adult vegetative shoots. In contrast, both free-base CKs declined in the fruiting phenological phase, and the IAA level increased dramatically. It was concluded that in phases characterized by intense organ formation, such as in the juvenile and flowering stages, there was an enhancement of CK content, mainly caused by zeatin, leading to a lower IAA/CK ratio. Higher ratios were correlated with phases that showed no organogenesis, such as adult and fruiting phenologies. Received April, 15, 1999; accepted September 7, 1999     

Influence of White Clover Mosaic Potexvirus Infection on the Endogenous Cytokinin Content of Bean

The cytokinin content in the primary leaves of bean (Phaseolus vulgaris) was monitored for 10 d after inoculation with white clover mosaic potexvirus. The cytokinins were isolated, purified, separated by high-performance liquid chromatography, and quantified by radioimmunoassay. The cytokinins detected at the time of inoculation (d 0) were: (a) the free bases, zeatin (Z), dihydrozeatin (DZ), and isopentenyladenine; (b) the riboside, DZ riboside (DZR); (c) the O-glucosides of DZ, DZR, and Z riboside; (d) the nucleotides, Z riboside-5′-monophosphate and isopentenyladenosine-5′-monophosphate; and (e) trace amounts of Z-9-glucoside and DZ-9-glucoside. During the 10 d after inoculation with white clover mosaic potexvirus, marked quantitative changes in this cytokinin profile were observed. The concentration of the free bases and DZR decreased, accompanied by an increase in the 9-glucosides and the nucleotides. Virus titer increased rapidly 3 d after inoculation, attaining a maximum level at d 5. This increase coincided with the increases in the 9-glucosides and the nucleotides. We propose that the decline in the cytokinin free bases and riboside may allow the increase of virus titer in bean and lead to the senescence of infected leaves.