Endogenous cytokinins in the vascular cambial region of Pinus sylvestris during activity and dormancy

Elucidation of the role of endogenous cytokinins in cambial activity and wood formation requires knowledge of their identity and concentrations in the cambial region. Here, we have used capillary liquid chromatography/frit-FAB mass spectrometry to identify endogenous cytokinins in the vascular cambial region of mature Pinus sylvestris (L.) trees. Full-scan mass spectra were obtained for isopentenyladenine, isopentenyladenosine, zeatin riboside, dihydrozeatin and dihydrozeatin riboside. Of these, isopentenyladenine, dihydrozeatin and dihydrozeatin riboside are demonstrated by rigorous physico chemical methods for the first time in a conifer. In addition, an adenine glycoside was found for the first time in a plant. The identified cytokinins were quantified in active and dormant cambial region tissues by isotope dilution techniques using the appropriate deuterated isotope for each cytokinin species. The concentration of the detected cytokinins ranged between 1.3 and 5.5 pmol g^-1 fresh weight, and did not vary greatly between dormant tissues, and in tissues actively dividing and differentiating. This observation indicates that cessation and reactivation of cell division activity in the vascular cambium is controlled by factors other than cytokinin availability.     

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 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.     

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.     

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.     

Glucosyl Zeatin and Glucosyl Ribosylzeatin from Vinca rosea L. Crown Gall Tumor Tissue

Recently detected but unidentified cytokinin activity in crown gall tumor tissue from Vinca rosea L. grown on media containing sources of reduced nitrogen has now been attributed to two adenine-type cytokinins. These compounds are glucopyranosyl derivatives of zeatin and ribosylzeatin. The substitution in each case is on the isopentenyl chain of the parent compound. Neither of these compounds had activity in the soybean callus bioassay at concentrations lower than 1 nm whereas zeatin had activity at 0.1 nm.     

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.     

Storage Organ Development in Radish (Raphanus sativus L.). 2. Effects of Growth Promoters on Cambial Activity in Cultured Roots, Decapitated Seedlings and Intact Plants

The radish varieties Cherry Belle and Long White Icicle wereused to investigate the role of the shoot and the effects ofsynthetic growth promoters in controlling cambial activity inthe seedling axis. Development was compared in excised roots, roots with hypocotylsattached and intact seedlings cultured aseptically on a nutrientmedium. No cambial divisions were seen in isolated radicleswhich had been cultured for ten days following excision butretention of hypocotyl tissue or the entire shoot resulted incambial activity and the production of secondary vascular tissues.Enriching the culture medium by raising the sucrose conantrationto 8% and including 10^–5 M indol-3yl acetic acid (IAA)5 x 10^–6 M 6-benzylaminopurine (BA) and 5 x 10^–4Minositol enhanced root thickening, increasing stele and xylemdiameters in roots cultured both with and without attached shoottissues. The effects of shoot tissues and enrichment of themedium were additive. The effects of auxin, cytokinin and gibberellin (gibberellicacid, GA²) were also studied on daxpitated seedlings. BA wasmuch more effective in inducing cell divisions in the hypocotylthan either IAA or GA supplied separately but a mixture of IAA+GAalso produced clearly defined arcs of cambial tissue. Littlesecondary tissue had been produced after seven days' treatment,and stelar enlargement was due to the development of a cambialzone and cell expansion in the primary tissues. Only minor differencesin response were observed between the two varieties. No stimulation of storage organ development occurred when auxin,cytokinin or inositol was inwrporated into the inorganic culturesolution in which plants of Cherry Belle were grown. Rnphanus sarivus, radish, storage organ, cambial activity, growth promoters, indol-3-ylacetic acid, 6-benzylaminopurine, gibberellic acid     

Regulators of Cell Division in Plant Tissues VUI. The Cytokinins of the Apple Fruit

The cytokinin activities of extracts of organs developed from the apple fruit bud were compared using the carrot phloem bioassay, and the identity of the cytokinins in the apple fruitlet was investigated. The activity of apple fruitlet extracts was slightly greater than the activity of pedicel extracts, and considerably greater than the activities of extracts of other organs. Extracts of the developing seeds of fruitlets were much more active than extracts of fruitlet flesh. Apple fruitlet extracts contained three principal cytokinins. One was identified as a 6-(substituted amino)purine and was either zeatin or some very closely related compound. The two other cytokinins exhibited the chromatographic behaviour of zeatin riboside and zeatin ribotide. A cytokinin extracted from vegetative apple shoots was chromatographically indistinguishable from zeatin.     

Cytokinins in seedling roots of pea

The natural occurrence of cytokinins existing both in a free form and as a constituent of transfer RNA was examined in serial segments of young seedling roots of pea. Purified ethanol extracts of root apices were resolved into four factors capable of inducing soybean callus tissue proliferation. The most active factor was identified as zeatin or some closely related compound; it produced polyploid divisions and tracheary element differentiation when tested on cultured pea root segments. The terminal 0- to 1-millimeter root tip contained 43 to 44 times more free cytokinin on a fresh weight or a per cell basis than the next 1- to 5-millimeter root segment. Extracts of more proximal segments behind the tip contained no measurable free cytokinin. Acid hydrolysates of transfer RNA exhibited reproducible cytokinin activity. Bioassays revealed that the predominant amounts of free cytokinin and that present in transfer RNA were restricted to the extreme root tip. There was approximately 27 times more free cytokinin than the amount detected in transfer RNA in root apices.     

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.     

Effects of Root Cutting on Cytokinin Content in the Shoot Apex Cells of Arabidopsis Plants

The dependence of cytokinin accumulation in the shoot apexes of Arabidopsis plants on the delivery of these hormones from the roots was studied. For the estimation of cytokinin content in the cells, the immunohistochemical localization method using antibodies against zeatin riboside was used. Differential conjugation of free cytokinin bases and their ribosides was used to prevent washing of cytokinins during the dehydration process. Root cutting decreased the immunostaining of zeatin in the cells of the shoot apical meristem, thereby supporting the hypothesis about dependence of cytokinin accumulation in these cells on the hormone delivery from the roots. The level of cytokinins in the cells of the shoot apex decreased under the effect of protonophore, indicating the important role of the secondary-active transmembrane transport process of cytokinins in the maintenance of their level in the cells of the shoot apex.     

Changes in growth-substance contents during growth of wheat grains

Samples of developing wheat grains were extracted at weekly intervals from ear emergence until maturity and the growth substances estimated by bioassay. Immature grains contained two cytokinins; one was similar to zeatin and another, with more cytokinin activity, had different properties. Ovules contained only small amounts of growth substances but at the end of anthesis the grains had a maximum content of cytokinin. The gibberellin content increased until 3 wk after anthesis then decreased; their auxin content increased until 4 wk after anthesis but decreased as the grains ripened and lost fresh weight. The husks contained smaller amounts of growth substances than the grains they surrounded. Exudates from young stems contained cytokinins and these may originate in the roots and move to the ears through the stems. The auxin in the grains was identified as indole-3yl-acetic acid and may be derived from the phenols present reacting with tryptophan.     

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.     

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.     

Exogenous zeatin accumulation in wheat root cells shows its role in regulation of cytokinin transport

Here we have shown that 24 hours after addition of zeatin to the nutrient solution the cytokinin content in xylem sap of wheat plants appears to be about 10 times lower that in the nutrient solution. Cytokinins accumulated mostly in roots and not in shoots of treated plants. These data demonstrate the existence of some barrier on cytokinin pathway from the nutrient solution to the plant shoot. With the help of Sudan III an increase in lignin and suberin deposition in the endodermis could be detected, being stronger with the increase in the distance from the root tip. The increase in deposition of suberin and lignin coincided with the decrease in cytokinin immunolabeling in root cells revealed with the help of monoclonal cytokinin antibodies and the second gold-labelled antibodies. Simultaneously exogenous cytokinins accumulated in root stele cells showing that the Casparian band was not only barrier on cytokinin pathway to plant shoot. It is concluded that high cytokinin immunolabe ling in the stele parenchyma cells around the stele vessels demonstrated accumulation of cytokinins by these cells, which could be important in regulation of cytokinin loading to the xylem vessels during there transport to the shoot. The role of cytokinin transporters is discussed.     

Quantification of the export of cytokinins from roots to shoots of Rosa hybrida and their degradation rate in the shoot

Cytokinins from the roots may be involved in regulating rose ( Rosa hybrida ) shoot growth and development. The objective of this study was to estimate the export of cytokinins from the roots and their degradation rate in the shoot, which were expected to be correlated with plant development. Hence, the total cytokinin content of the shoot, the concentration of zeatin riboside (ZR) in bleeding sap, and the transpiration rates in three stages of development were determined. The estimations performed are based on the assumption that the cytokinin concentration in bleeding sap is representative for the cytokinin concentration in xylem sap in situ. This was verified by comparing the ZR concentration in bleeding sap and in sap obtaíned after pressurizing the root system to a level equivalent to the leaf water potential; no significant differences could be found. The import of cytokinins could not be correlated with plant development, as it increased linearly with time. The estimated relative degradation rate of cytokinins in the shoot decreased as the plants matured. The half-life of cytokinins in the shoot was found to be approximately 1 day, indicating that cytokinins are rapidly metabolized in the shoot.     

Comparison of effects of bacterial strains differing in their ability to synthesize cytokinins on growth and cytokinin content in wheat plants

The content of cytokinins and pigments together with the morphological parameters and fresh weight were estimated in durum wheat (Triticum durum Desf.) plants 2–4 days after introduction into their rhizosphere of an aliquot of Bacillus suspension using the strains that differed in their ability of producing cytokinins. The experiments were performed under laboratory conditions at the optimum light intensity and mineral nutrition. Inoculation with microorganisms incapable to synthesize cytokinins did not affect the total cytokinin content in the wheat plants, whereas the presence of cytokinin-producing microorganisms in the rhizosphere was accompanied by a considerable increase in the total cytokinin content and the accumulation of individual hormones. On the second day after inoculation, a dramatic increase in zeatin riboside and zeatin O-glucoside contents was observed in the roots, and at the next day the accumulation of zeatin riboside and zeatin was registered in the shoots of treated plants. The increase in cytokinin content promoted plant growth (the increased leaf length and width and a faster accumulation of plant fresh and dry weight). Plant treatment with a substance obtained from microorganisms incapable to synthesize hormones resulted in the insignificant growth stimulation. Plant treatment with a substance obtained from cytokinin-producing microorganisms increased leaf chlorophyll content; in this case, the level of chlorophylls was comparable to that observed in the plants treated with a synthetic cytokinin benzyladenine. The role of cytokinins of microbial origin as a factor providing for growth-stimulating effect of bacteria on plants is discussed.     

Cytokinins in developing fruits of Moringa pterigosperma Gaertn.

The existence of cytokinins both as a free form and as a constituent of t-RNA was investigated in young fruits of Moringa pterigosperma Gaertn. Purified methanol extract was separated into butanol insoluble and butanol soluble fractions. The cytokinin(s) in the butanol insoluble fraction was tentatively identified as zeatin nucleotide. The butanol soluble fraction contained cytokinins and was chromatographed on Sephadex LH-20 with 35% ethanol. The two active fractions from LH-20 column coincided with zeatin and zeatin riboside. Cytokinin per g tissue was high in early stages of fruit growth and then remained more or less constant. Alkaline phosphatase hydrolysis of t-RNA hydrolysate of fruit tissue showed considerable cytokinin activity.