Cytokinin Activity in Lupinus albus

The cytokinin content of the root exudate and leaves of fruiting white lupin plants (Lupinus albus L.) was investigated at 2 weekly intervals after anthesis of the lowest flower on the primary inflorescence. Up to 8 weeks after anthesis the level of cytokinins in the root exudate increased. However, at 10 weeks after anthesis insufficient sap was produced for analysis. Cytokinins co-eluting with zeatin and zeatin riboside were detected in the root exudate after fractionation on Sephadex LH-20. The cytokinin levels in the mature leaves steadily increased up to 8 weeks after anthesis and thereafter remained relatively constant. Three peaks of activity, co-eluting with zeatin, zeatin riboside and the glucoside cytokinins were recorded in the leaf extracts. The level of glucoside cytokinins in the leaves was high at 8 and 10 weeks after anthesis. Paper chromatography of extracts of fruits collected at 2 weeks after anthesis indicated that as fruit development proceeded there was a build up of cytokinin in this region of the plant. It is suggested that, in the white lupin, the cytokinins translocated to the shoot are accumulated in the leaves and in the fruits and that it is only later when there is a considerable decrease in sap (10 weeks after anthesis) production that a decreasing supply of cytokinins leads to shoot senescence.     

Ontogenetic variation of four cytokinins in soybean root pressure exudate

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

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

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

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

     

Changes in Cytokinin Activity during Seed Germination in Rice (Oryza sativa L.)

Cytokinin activity was determined in dry mature rice seeds,in endosperm and embryo tissues 24, 48 and 72 over imbibitionand in radicles 96 h after germination. Cytokinins with chromatographicproperties similar to zeatin, zeatin riboside, zeatin glucosideand zeatin riboside glucoside were datected in embryo and endosperm,but only the latter two were detected in mature seeds. Cytokininactivity was low during early toges of germination. Qualitativeand quantitative changes in cytokinins were observed in bothembryo and endosperm. The presence of higher cytokinin activityin the endosperm than in the embryo during the first 24 h aftergermination suggests that the endosperm may supply cytokininsuntil the embryo is able to synthaize its own cytokinins. Thepossible significance of high cytokinin glucoside activity inthe embryo early during germination and high cytokinin activityin the radicle during the later stages is discussed. Oryza sativa L., rice, cytokinin, germination, seed     

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.     

The Effect of Photoperiod on Levels of Endogenous Cytokinins in Xanthium strumarium

The cytokinin content of Xanthium strumarium L. plants decreased markedly when they were exposed to short days (SD). There was a significant decrease in the content of the butanol-soluble cytokinins of the mature leaves after only 5 SD cycles, and after 10 SD there was no significant cytokinin activity in butanol extracts; the changes in the young leaves were less marked. Most of the cytokinin activity in mature leaves appears to be present in the aqueous fraction, whereas in young leaves most activity occurs in the butanol-soluble fraction. SD treated plants produced less root exudate than LD plants, but there were no significant differences in the amounts of cytokinin in the root exudates from LD and SD plants collected over an equivalent time period. The cytokinin levels of SD-induced leaves remained low even when transferred back to LD. The observed differences in cytokinin levels did not appear to be the result of photosynthetic differences. Exposure of detached leaves to LD or SD did not result in differences in cytokinin content. It is not clear whether the observed changes in cytokinin levels in the leaves under SD are involved in the flowering response, but they may be causally related to a reduced chlorophyll content observed in SD-induced leaves.     

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.     

A Comparison of the Endogenous Cytokinins in the Roots and Xylem Exudate of Nematode-resistant and Susceptible Tomato Cultivars

Non-infested roote of a tomato cultivar (Roodeplaat Premier)susceptible to root-knot nema-todes contained higher levelsof endogenous cytokinins than non-infested roots of a nematode-resistantcultivar (Master Fl). In both cultivars nematode infestationincreased the cytokinin level in the roots. Sephadex LH-20 fractionationindicated that the xylem exudate of infested susceptible plantscontained zeatin and zeatin riboside. In the roots of both cultivarsa third compound, which co-eluted with zeatin glucoside, waspresent. The activity of all three cell division-inducing compoundswas higher in the roots of susceptible plants than in rootsof nematode-resistant plants. The present results support thehypothesis that cytokinins may be a controlling factor in thedevelopment of giant cells.     

A Cytokinin Complex in the Developing Fruits of Lupinus albus

The apices of Lupinus albus L. were analysed for cytokinin activity at three stages of development. Little cytokinin activity could be detected in the apices at the time of flowering. However, a considerable amount of activity was detected as the fruits developed. Separate analyses of seed and pod material indicated that there was a high level of cytokinin in both these parts of the fruit. After fractionation of the peaks of activity obtained from paper chromatograms on Sephadex LH-20, four peaks of cytokinin activity were recorded. Two of these co-eluted with zeatin and zeatin riboside. A third peak at an elution volume of 360–440 ml could be hydrolysed with β-glucosidase to give activity at elution volumes corresponding to those of zeatin and zeatin riboside. This strongly suggested that both glucosylated zeatin and glucosylated zeatin riboside were present in the developing fruits of L. albus. The fourth peak at an elution volume of 160–280 ml did not disappear upon hydrolysis with β-glucosidase, and it is possible that it represented a nucleotide cytokinin.     

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.     

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.     

Cytokinins in Populus x robusta Schneid: A complex in leaves

Summary At least seven cytokinins have been detected in mature leaves of Populus x robusta Schneid after chromatography on Sephadex LH-20. Two of these have similar elution volumes to zeatin and zeatin riboside. A third appears to be a cytokinin glucoside. A fourth is a new, unidentified cytokinin, susceptible to mild oxidation, and yielding two cytokinin active products after acid hydrolysis. This cytokinin complex has been found in fully expanded leaves, a tissue in which cell division is completed.     

Hormones in Plants Bearing Nitrogen-fixing Root Nodules: Distribution and Seasonal Changes in Levels of Cytokinins in Alnus glutinosa (L.) Gaertn

The root nodules of both dormant and non-dormant plants of Alnusglutinosa (L.) Gaertn. were found (by the soybean callus bioassay)to contain levels of cytokinin activity greatly exceeding thoseof other parts of young vegetative plants. A large, transient increase in cytokinin activity occurred inthe nodules at the time of bud break. Similar, although muchsmaller, increases were detected also in roots and buds. Theincrease in the level of nodule cytokinin activity was observedboth in mature trees and in young pot-grown plants in two successiveyears. A second peak of cytokinin activity, considered to bederived from cytokinin nucleotides, was found in the nodulesof mature trees in midsummer. Analysis of cytokinin extracts of different plant parts by meansof a Sephadex LH20 column revealed the presence of three mainpeaks of activity, with elution volumes corresponding to thoseof zeatin-9-glucoside, zeatin riboside, and zeatin. While theglucoside-like peak -was predominant in the nodules and leavesit was not detected in root pressure sap. A zeatin ribo-side-likepeak was the major cytokinin in the roots and root pressuresap. These findings are discussed in relation to current hypothesesconcerning the production, distribution, and possible physiologicalroles of the cytokinins.     

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.     

Alterations of Endogenous Cytokinins in Transgenic Plants Using a Chimeric Isopentenyl Transferase Gene

Cytokinins, a class of phytohormones, appear to play an important role in the processes of plant development. We genetically engineered the Agrobacterium tumefaciens isopentenyl transferase gene, placing it under control of a heat-inducible promoter (maize hsp70). The chimeric hsp70 isopentenyl transferase gene was transferred to tobacco and Arabidopsis plants. Heat induction of transgenic plants caused the isopentenyl transferase mRNA to accumulate and increased the level of zeatin 52-fold, zeatin riboside 23-fold, and zeatin riboside 5[prime]-monophosphate twofold. At the control temperature zeatin riboside and zeatin riboside 5[prime]-monophosphate in transgenic plants accumulated to levels 3 and 7 times, respectively, over levels in wild-type plants. This uninduced cytokinin increase affected various aspects of development. In tobacco, these effects included release of axillary buds, reduced stem and leaf area, and an underdeveloped root system. In Arabidopsis, reduction of root growth was also found. However, neither tobacco nor Arabidopsis transgenic plants showed any differences relative to wild-type plants in time of flowering. Unexpectedly, heat induction of cytokinins in transgenic plants produced no changes beyond those seen in the uninduced state. The lack of effect from heat-induced increases could be a result of the transient increases in cytokinin levels, direct or indirect induction of negating factor(s), or lack of a corresponding level of competent cellular factors. Overall, the effects of the increased levels of endogenous cytokinins in non-heat-shocked transgenic plants seemed to be confined to aspects of growth rather than differentiation. Since no alterations in the programmed differentiation pattern were found with increased cytokinin levels, this process may be controlled by components other than absolute cytokinin levels.     

Cytokinin biochemistry in relation to leaf senescence. VIII. Translocation, metabolism and biosynthesis of cytokinins in relation to sequential leaf senescence of tobacco

Cytokinin bases (zeatin and dihydrozeatin) and ribosides (zeatin riboside and dihydrozeatin riboside) were identified as major cytokinins in tobacco xylem sap by radioimmunoassay. When ³H-labelled zeatin riboside or dihydrozeatin riboside were supplied to tobacco plants via the xylem, leaves of differing maturity did not differ appreciably in level of radioactivity or in metabolism of the cytokinin. The major metabolites of zeatin riboside in leaves were adenine, adenosine and adenine nucleotides, whereas that of dihydrozeatin riboside was dihydrozeatin 7-glucoside. Incorporation of [¹⁴C]adenine into zeatin was evident in upper green leaves. indicating that young leaves have the capacity to synthesize cytokinins in situ. In contrast, fully expanded green leaves and senescing tobacco leaves exhibited little or no incorporation of [¹⁴C]adenine into cytokinins. This difference in cytokinin biosynthetic capacity may contribute to the differing cytokinin levels in leaves of different matirity, and may participate in control of sequential leaf senescence in tobacco.     

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.     

Changes in cytokinin levels of Phalaenopsis leaves at high temperature

The effect of high temperatures on cytokinin levels in Phalaenopsis hybrida leaves was investigated. Endogenous cytokinins were identified and quantified in Phalaenopsis leaves grown under high temperature conditions (30/25 °C day/night) using high performance liquid chromatography, bioassay and gas chromatography-selected ion monitoring-mass spectrometry. After 5 and 20 d of low temperature (25/20 °C day/night), zeatin, zeatin riboside and dihydrozeatin levels in the leaves were higher than that in leaves subjected to high temperature treatments. When Phalaenopsis leaves were exposed to low temperatures, about 76 % of the free cytokinins detected were of the zeatin-type. Glucoside cytokinins in the leaves increased significantly 5 d following high temperatures, and the rate of increase in glucoside cytokinins corresponded to the duration of high temperatures. At the same time, zeatin riboside and dihydrozeatin declined significantly following high temperature application. A significant accumulation of glucoside cytokinins, zeatin-9-glucoside, zeatin-O-glucoside, zeatin riboside-O-glucoside, and dihydrozeatin-O-glucoside was observed 20 d following high temperatures. These results suggest that high temperatures lead to an accumulation of glucoside cytokinins and a reduction of free base and riboside cytokinins.     

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.     

Dependence of cytokinin distribution in plants on their physical and chemical properties and transpiration rate

The effect of transpiration on cytokinin accumulation and distribution in 7-day-old wheat (Triticum durum Desf.) seedlings grown on nutrient medium supplemented with zeatin or its riboside was studied. The content of cytokinins in plants and nutrient medium was measured by the immunoenzyme analysis; cytokinin distribution between root cells was assessed immunohistochemically using antibodies against zeatin derivatives. The rate of transpiration was reduced 20-fold by plant placing in humid chamber. At normal transpiration, after 6 h of plant incubation on the solution of zeatin, the level of cytokinins in plant tissues increased stronger than after incubation on the solution of zeatin riboside (by 7.3 and 3.5 times, respectively, as compared with control), although the rates of both cytokinin uptake were equal. Most portions of cytokinins were retained in the roots, which was stronger expressed in the case of free zeatin uptake. A decrease in the rate of transpiration did not affect substantially the zeatin absorption from nutrient medium and the total level of cytokinin accumulation in plants, but these indices were sharply decreased in the case of zeatin riboside. In the zone of absorption of both control roots and roots treated with cytokinins, more intense cytokinin immunostaining was observed in the cells of the central cylinder. The interrelation between cytokinin distribution between the cells and apoplast, their inactivation, and transport over the plant and their form (zeatin or zeatin riboside) used for treatment is discussed.     

The Effect of Ringing on Cytokinin Distribution in Salix baylonica

Although quantitative differences were observed in the cytokinin content of mature leaves and bark of Salix babylonica it would appear as if these tissues contained the same cytokinin complement. Ringing resulted in a decrease in the level of cytokinins in the leaves and an increase in the bark, both above and below the girdle. In the leaves the decrease was due mainly to a drop in the level of those compounds that co-chromatographed with the cytokinin glucosides. These compounds were also almost undetectable in the bark above the girdle, where callus was formed. The observed increase in the cytokinin content of the bark above the girdle was due to higher activity in those parts of the chromatograms where zeatin and zeatin riboside occurred. Ringing stimulated the growth of lateral buds below the girdle. These developing buds as well as the bark below the girdle contained very high levels of cytokinins that cochromatographed with zeatin and zeatin riboside.