Changes in Concentrations of Cytokinins (CKs) in Root and Axillary Bud Tissue of Miniature Rose Suggest that Local CK Biosynthesis and Zeatin-Type CKs Play Important Roles in Axillary Bud Growth

Involvement of cytokinins (CKs) in axillary bud growth of miniature rose was studied. Variation in root formation and axillary bud growth was induced by two indole 3-butyric acid (IBA) pretreatments in two cutting sizes. At six physiological developmental stages around the onset of axillary bud growth, concentrations of CKs were determined in both root and axillary bud tissue by liquid chromatography combined with electrospray tandem mass spectrometry (LC-ESP-MS/MS). Chronological early onset of axillary bud growth occurred in long cuttings pretreated at low IBA concentration, whereas physiological early root formation was associated with long cuttings and high IBA concentration. The CKs zeatin (Z), isopentenyl adenine (iP), zeatin riboside (ZR), dihydrozeatin riboside (DHZR), isopentenyl adenosine (iPA), zeatin O-glucoside (ZOG), zeatin riboside O-glucoside (ZROG), zeatin riboside 5′-monophosphate (ZRMP), and isopentenyl adenosine 5′-monophosphate (iPAMP) were detected. Concentrations of CKs in axillary bud tissue far exceeded those in root tissue. Indole 3-butyric acid pretreatment influenced the concentration of CKs in axillary bud tissue more than did cutting size, whereas pretreatments only slightly affected CKs in root tissue. The dominant CKs found were iPAMP and ZR. An early and large increase in iPAMP indicated rapid CK biosynthesis in rootless cuttings, suggesting that green parts, including the axillary bud, can synthesize CKs. At the onset of axillary bud growth an increase in concentration of Z, ZR, ZRMP, ZOG, and ZROG was largely coincident with a decrease in iPAMP, iPA, iP, and DHZR. After the onset of axillary bud growth, CK content largely decreased. These results strongly indicate a positive role for CKs in axillary bud growth, and presumably ZRMP, ZR, and Z are active in miniature rose.     

Rapid increases in cytokinin concentration in lateral buds of chickpea (Cicer arietinum L.) during release of apical dominance

We examined the role of cytokinins (CKs) in release of apical dominance in lateral buds of chickpea (Cicer arietinum L.). Shoot decapitation or application of CKs (benzyladenine, zeatin or dihydrozeatin) stimulated rapid bud growth. Time-lapse video recording revealed growth initiation within 2 h of application of 200 pmol benzyladenine or within 3 h of decapitation. Endogenous CK content in buds changed little in the first 2 h after shoot decapitation, but significantly increased by 6 h, somewhat later than the initiation of bud growth. The main elevated CK was zeatin riboside, whose content per bud increased 7-fold by 6 h and 25-fold by 24 h. Lesser changes were found in amounts of zeatin and isopentenyl adenine CKs. We have yet to distinguish whether these CKs are imported from the roots via the xylem stream or are synthesised in situ in the buds, but CKs may be part of an endogenous signal involved in lateral bud growth stimulation following shoot decapitation. To our knowledge, this is the first detailed report of CK levels in buds themselves during release of apical dominance. Received: 12 December 1996 / Accepted: 7 January 1997     

Hormone Levels and Apical Dominance in the Aquatic Fern Marsilea drummondii A. Br

Terminal buds and successively subjacent lateral buds of the water fern, Marsilea drummondii, were examined to determine the pattern of hormone distribution in relation to apical dominance. Quantitative levels of indole-3-acetic acid (IAA), abscisic acid (ABA), zeatin and zeatin riboside (Z and ZR), and isopentenyladenosine (iPA) were determined by a solid-phase immunoassay using polycional antihormone antibodies. Enzyme-linked immunosorbent assay was used following a one-step HPLC purification procedure to obtain the free hormones. Active shoot apices contained the most IAA and Z-type cytokinins and inhibited buds the least. No significant differences in ABA levels were found leading to the conclusion that ABA did not play any role in apical dominance. The normal precedence of the most rapid outgrowth of the youngest inhibited bud as observed previously in decapitated plants was well correlated with its very high level of iPA observed in this study. The same phenomenon was observed in the median buds but with a weaker amplitude. The presence of this storage form could indicate that a bud at its entry into quiescence eventually looses the ability to hydroxylate iPA to Z-type cytokinins when it is fully inhibited. IAA and Z + ZR are concluded to be essential for lateral bud growth.     

Transport and metabolism of xylem cytokinins during lateral bud release in decapitated chickpea (Cicer arietinum) seedlings

Although cytokinins (CKs) are widely thought to have a role in promoting shoot branching, there is little data supporting a causative or even a correlative relationship between endogenous CKs and timing of bud outgrowth. We previously showed that lateral bud CK content increased rapidly following shoot decapitation. However, it is not known whether roots are the source of this CK. Here, we have used shoot decapitation to instantaneously induce lateral bud release in chickpea seedlings. This treatment rapidly alters rate and direction of solvent and solute (including CK) trafficking, which may be a passive signalling mechanism central to initiation of lateral bud release. To evaluate changes in xylem transport, intact and decapitated plants were infiltrated with [³H]zeatin riboside ([³H]ZR), a water‐soluble blue dye or [³H]H₂O by injection into the hypocotyl. All three tracers were recovered in virtually all parts of the shoot within 1 h of injection. In intact plants, solute accumulation in the lateral bud at node 1 was significantly less than in the adjacent stipule and nodal tissue. In decapitated plants, accumulation of [³H]ZR and of blue dye in the same bud position was increased 3‐ to 10‐fold relative to intact plants, whereas content of [³H]H₂O was greatly reduced indicating an increased solvent throughput. The stipule and cut stem, predicted to have high evapotranspiration rates, also showed increased solute content accompanied by enhanced depletion of [³H]H₂O. To assess whether metabolism modifies quantities of active CK reaching the buds, we followed the metabolic fate of [³H]ZR injected at physiological concentrations. Within 1 h, 80–95% of [³H]ZR was converted to other active CKs (mainly zeatin riboside‐5′phosphate (ZRMP) and zeatin (Z)), other significant, but unconfirmed metabolites some of which may be active (O‐acetylZR, O‐acetylZRMP and a compound correlated with sites of high CK‐concentrations) and inactive catabolites (adenosine, adenine, 5′AMP and water). Despite rapid metabolic degradation, the total active label, which was indicative of CK concentration in buds, increased rapidly following decapitation. It can be inferred that xylem sap CKs represent one source of active CKs appearing in lateral buds after shoot decapitation.     

Changes in Cytokinins and Gibberellin-Like Substances in Pinus radiata Buds during Lateral Shoot Initiation and the Characterization of Ribosyl Zeatin and a Novel Ribosyl Zeatin Glycoside

Based on detection and quantitation by bioassay, endogenous gibberellin-like substances (GAs) and cytokinins (CKs) in Pinus radiata D. Don buds during sequential shoot initiation shift from less polar to more polar forms (GAs) and from conjugated to free forms (CKs). As the terminal bud moves from the production of “short shoots” (needle fascicles) to “long shoots” (lateral branches or female conebuds), a more polar GA appears while a glucoside-conjugate of zeatin riboside is reduced, and zeatin riboside levels increase markedly.     

Cytokinins and bud morphology in Pinus radiata

Cytokinins (CKs) play essential roles in the regulation of plant growth and development. In the previous paper (Zhang et al. 2001), we reported the detection and identification of a wide spectrum of CKs, including several novel forms, in the buds of Pinus radiata D. Don. In this paper we examine the relationship between the CKs and buds from juvenile and adult trees of P. radiata. During development the morphology of buds alters significantly, from buds bearing primary needles during their juvenile phase to buds sealed in scales at the adult phase. The morphology of adult buds is a very stable character, as fascicle meristems released from apical dominance, or cultured in vitro, produced only secondary needles. However, exogenous CK causes the adult buds to revert to juvenile bud development in vitro . Analyses of the endogenous CKs revealed that juvenile buds had a relatively higher level of isopentenyladenine and isopentenyladenosine, extremely low levels of phosphorylated CKs and a relatively low level of novel CK glycosides. The adult buds contained lower levels of free base and riboside CKs but very high levels of phosphorylated CKs and novel CK glycosides. Possible roles for CKs in the regulation of bud development are discussed.     

The control of bud dormancy in potato tubers. Measurement of the seasonal pattern of changing concentrations of zeatin-cytokinins

A radioimmunoassay, combined with high-performance liquid chromatography, has been used to analyse the zeatin-type cytokinins of potato (Solanum tuberosum L. cv. Majestic) tubers and tuber buds throughout growth and storage. During tuber growth, zeatin riboside was the predominant cytokinin detected in all tissues. Immediately after harvest, the total cytokinin concentration fell dramatically in the storage tissue, largely as a consequence of the disappearance of zeatin riboside. During storage, levels of cytokinins in the storage tissue remained relatively constant, but increased in the tuber buds. In the buds of tubers stored at 2°C there was a 20-to 50-fold increase in total cytokinin over six weeks, coinciding with the natural break of innate dormancy. At 10°C the rise in the level of bud cytokinins was slower, correlating with the longer duration of innate dormancy. Injecting unlabelled cytokinins into tubers in amounts known to induce sprouting gave rise to increases in cytokinin concentrations in the buds of the same order as the increase associated with the natural break of dormancy. Metabolism of injected cytokinins was greater in non-dormant than in dormant tubers. The roles of cytokinin concentration and the sensitivity of the buds to cytokinin in the control of dormancy are discussed.Abbreviations CK cytokinin - FW fresh weight - HPLC high-performance liquid chromatography - RIA radioimmunoassay - tio⁶ade 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-purine=zeatin - tio⁶adeglc⁹ 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-glucopyranosyl purine=zeatin-9-glucoside - tio⁶ado 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-ribofuranosyl purine=zeatin riboside - tio⁶ado-[³H]-diol a radioactive derivative of zeatin riboside, synthesised by periodate-oxidation followed by [³H]NaBH₄-reduction - tio⁶AMP 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-5-phosphoribofuranosyl purine=zeatin riboside 5-monophosphate - t(ioglc⁴)⁶ade 6-(4-O--D-glucopyranosyl-3-methylbut-trans-2-enylamino)-purine=zeatin-O-glucoside     

Cytokinins from terminal buds of Euphoria longana during different growth stages

The presence of endogenous cytokinins were detected in the terminal buds of longan ( Euphoria longana Lam.) after purification by ion exchange and Sephadex LH-20 chromatography, and bioassay, enzymic degradation, high-performance liquid chromatography and gas chromatography-mass spectrometry. Permethylated derivatives of two highly active cytokinin glucoside compounds from dormant buds were: 6-(4-O-β-D-glucosyl-3-methyl-but-2-enylamino) purine (zeatin-O-glucoside) and 9-β-D-ribofuranosyl-6-(4-hydroxy-3-methyl-but-2-enylamino) purine (zeatin riboside-O-glucoside). Simultaneously, four active cytokinins from buds at the stages of leaf flush and flower bud initiation were identified as 6-(4-hydroxy-3-methyl-but- trans -2-enylamino) purine (zeatin), zeatin-9-β-D-ribofuranosylpurine (zeatin riboside), 6-(3-methyl-2-butenyl) aminopurine (isopentenyladenosine, 2iPA) and N-(3-methyl-2-butenyl) adenine (isopentenyladenine, 2iP). The total cytokinin levels were low at leaf flush, with the zeatin and zeatin riboside in the buds about 70% of the total. In the transition of the terminal bud from leaf flush to dormancy, the principal cytokinins were zeatin-O-glucoside and zeatin riboside-O-glucoside. However, significant decreases in the levels of zeatin-O-glucoside and zeatin riboside-O-glucoside and increases in those of zeatin, zeatin riboside, 2iPA and 2iP were observed at flower bud initiation. It is suggested that in longan, the cytokinins that are translocated to the shoots are accumulated in the buds at the dormant stage, and that later there is a considerable increase in free cytokinins during flower bud initiation, leading to the promotion of flower bud development.     

Endogenous levels of abscisic acid, indole-3-acetic acid, zeatin and zeatin-riboside during the course of adventitious root formation in cuttings of Craigella and Craigella lateral suppressor tomatoes

Indole acetic acid (IAA). abscisic acid (ABA), and zeatin plus zeatin riboside (Z + ZR) were determined daily in cuttings of Lycopersicon esculentum Mill. cv. Craigella (C) and the Craigella Lateral Suppressor (CLS) mutant during the first 5 days of the root-forming process. A solid-phase enzyme immunoassay using specific anti-hormone antibodies was used following a one-step HPLC purification procedure. The hormone measurements were made in cuttings divided into 4 parts. The main variations occurred in the terminal bud and in the basal part of the hypocotyl of the two tomato varieties, i.e. significant IAA and ABA increases during the first 2 days followed by a more or less fast return to the initial values at day 4 or 5. This is probably due to the ablation of the root system. Z + ZR levels dramatically decreased in the basal part of the Craigella hypocotyl 1 day after cutting. Contrary to CLS, C hypocotyls recovered the initial high levels when roots regenerated (day 5). This is probably linked to the greater ability of roots to produce cytokinins in C plants than in CLS ones. The first step of root formation (reactivation of the pericyclic cells and formation of root primordia) corresponded to a high IAA/Z + ZR ratio in the root-forming tissue, whereas the second step (elongation of young roots) was characterized by a low ratio with low levels of hormones.     

Cytokinins in Vegetative and Reproductive Buds of Pseudotsuga menziesii

Immunoaffinity techniques using columns of immobilized antibodies raised against zeatin riboside and isopentenyladenosine were found to be effective in isolating cytoklnins from vegetative, female, and male buds of Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). The purified cytokinins were separated by reverse phase high performance liquid chromatography and analyzed by radioimmunoassay. Confirmation of cytokinin identities was by gas chromatography-mass spectrometry. Immediately prior to bud burst, all bud types contained three major cytokinins: isopentenyladenosine, zeatin riboside, and a hexose conjugate of zeatin riboside (not zeatin riboside O-glucoside). Zeatin-type cytokinins were present in relatively high concentration in vegetative and female buds. In male buds, however, relatively high levels of isopentenyladenosine were found together with low levels of zeatin-type cytokinins.     

Investigations into the Possibility that Potato Buds Synthesize Cytokinins

In an attempt to determine whether potato buds synthesize cytokinins,tubers and potato pieces were subjected to conditions whichboth retard and accelerate bud development. Cytokinin activitywas recorded in the tubers and sprouts under different experimentalconditions. Most of the compounds detected had chromatographicproperties resembling those of zeatin, zeatin riboside, andzeatin glucoside. It would appear as if the buds and sproutsdo not synthesize cytokinins. Initial bud growth may be dependenton the supply of cytokinins within the mother tubers, whilethe increase in the sprouts could be the result of root developmentat their basal ends.     

Changes in Cytokinins before and during Early Flower Bud Differentiation in Lychee (Litchi chinensis Sonn.)

Lychee (Litchi chinensis) has been analyzed for cytokinins in buds before and after flower bud differentiation, using reversephase high performance liquid chromatography in combination with Amaranthus bioassay and gas chromatography-mass spectrometry-selected ion monitoring. Four cytokinins, zeatin, zeatin riboside, N⁶-(δ²-isopentenyl)adenine, and N⁶-(δ⁶-isopentenyl) adenine riboside, were detected in buds. There was an increase of cytokinin activity in the buds during flower bud differentiation. In dormant buds, the endogenous cytokinin content was low, and the buds did not respond to exogenous cytokinin application. Application of kinetin promotes flower bud differentiation significantly after bud dormancy. These results are interpreted as an indication that the increase in endogenous cytokinin levels during flower bud differentiation may be correlative rather than the cause of flower bud initiation.     

Spatial and temporal changes in multiple hormone groups during lateral bud release shortly following apex decapitation of chickpea (Cicer arietinum) seedlings

Although the co-ordination of promotive root-sourced cytokinin (CK) and inhibitory shoot apex-sourced auxin (IAA) is central to all current models on lateral bud dormancy release, control by those hormones alone has appeared inadequate in many studies. Thus it was hypothesized that the IAA : CK model is the central control but that it must be considered within the relevant timeframe leading to lateral bud release and against a backdrop of interactions with other hormone groups. Therefore, IAA and a wide survey of cytokinins (CKs), were examined along with abscisic acid (ABA) and polyamines (PAs) in released buds, tissue surrounding buds and xylem sap at 1 and 4 h after apex removal, when lateral buds of chickpea are known to break dormancy. Three potential lateral bud growth inhibitors, IAA, ABA and cis -zeatin 9-riboside (ZR), declined sharply in the released buds and xylem following decapitation. This is in contrast to potential dormancy breaking CKs like trans -ZR and trans -zeantin 9-riboside 5'phosphate (ZRMP), which represented the strongest correlative changes by increasing 3.5-fold in xylem sap and 22-fold in buds. PAs had not changed significantly in buds or other tissues after 4 h, so they were not directly involved in the breaking of bud dormancy. Results from the xylem and surrounding tissues indicated that bud CK increases resulted from a combination synthesis in the bud and selective loading of CK nucleotides into the xylem from the root.     

Cytokinin translocation: Changes in zeatin and zeatin-riboside levels in the root exudate of tomato plants during their development

Summary The zeatin and zeatin riboside content of tomato (Lycopersicon esculentum Mill.) root exudates were determined at different stages of development. Zeatin riboside was found to be the major translocational form of cytokinin in the xylem during early vegetative growth. During flower bud formation this cytokinin decreased markedly in concentration so that, at anthesis, there was no appreciable difference in the zeatin and zeatin riboside concentration in the root exudate.     

The role of cytokinins in relation to flower-bud blasting in Iris cv. Ideal: Cytokinin determination by an improved enzyme-linked immunosorbent assay

The effect of dark and light treatment on endogenous cytokinins in internodes and buds of Iris was determined. Plant material was purified by chromatographic methods and cytokinins were assayed by an immunoassay.An indirect competitive enzyme immunoassay for the determination of zeatin- and isopentenyl-adenine cytokinins was developed. This assay, which is not dependent on the titre of the antibodies raised against zeatin riboside and isopentenyl-adenosine appeared to be specific, highly sensitive and more reproducible compared to a direct competitive enzyme immunoassay for cytokinins.Isopentenyl-adenosine was the most abundant cytokinin found, followed by zeatin: the latter counteracts bud blast when injected into dark-treated plants. Smaller amounts of isopentenyl-adenine and zeatin riboside were found. Results are in agreement with the hypothesis that deficiency of growth substances like cytokinins plays an important role in the occurrence of flower-bud blasting.A possible role for the major endogenous cytokinin, isopentenyl-adenosine, which earlier was found not to be effective in counteracting bud blast when injected into buds of dark-treated plants, is discussed.     

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.     

Correlation of xylem sap cytokinin levels with monocarpic senescence in soybean

Cytokinins (CKs) coming from the roots via the xylem are known to delay leaf senescence, and their decline may be important in the senescence of soybean (Glycine max) plants during pod development (monocarpic senescence). Therefore, using radioimmunoassay of highly purified CKs, we quantified the zeatin (Z), zeatin riboside (ZR), the dihydro derivatives (DZ, DZR), the O-glucosides, and DZ nucleotide in xylem sap collected from root stocks under pressure at various stages of pod development. Z, ZR, DZ, and DZR dropped sharply during early pod development to levels below those expected to retard senescence. Pod removal at full extension, which delayed leaf senescence, caused an increase in xylem sap CKs (particularly ZR and DZR), while depodding at late podfill, which did not delay senescence, likewise did not increase the CK levels greatly. The levels of the O-glucosides and the DZ nucleotide were relatively low, and they showed less change with senescence or depodding. The differences in the responses of individual CKs to senescence and depodding suggest differences in their metabolism. Judging from their activity, concentrations and response to depodding, DZR and ZR may be the most important senescence retardants in soybean xylem sap. These data also suggest that the pods can depress CK production by the roots at an early stage and this decrease in CK production is required for monocarpic senescence in soybean.     

The control of bud dormancy in potato tubers

Potato (Solanum tuberosum L.) tuber buds normally remain dormant through the growing season until several weeks after harvest. In the cultivar Majestic, this innate dormancy persisted for 9 to 12 weeks in storage at 10° C, but only 3 to 4 weeks when the tubers were stored at 2° C. At certain stages, supplying cytokinins to tubers with innately dormant buds induced sprout growth within 2 d. The growth rate was comparable to that of buds whose innate dormancy had been lost naturally. Cytokinin-treatment did not accelerate the rates of cell division and cell expansion in buds whose innate dormancy had already broken naturally. Gibberellic acid did not induce sprout growth in buds with innate dormancy. We conclude that cytokinins may well be the primary factor in the switch from innate dormancy to the non-dormant state in potato tuber buds, but probably do not control the subsequent sprout growth.Abbreviations tio ⁶ade 6-(4-hydroxy-3-methylbut-trans-2-enyl amino)purine, zeatin - tio⁶ado 6-(4-hydroxy-3-methylbut-trans-2-enyl amino)-9--D-ribofuranosyl purine, zeatin riboside     

Increases in cytokinin levels in Scots pine in relation to chilling and bud burst

Levels of isopentenyladenosine and zeatin riboside were monitored in buds and needles of Scots pine ( Pinus sylvestris L.) seedlings grown under controlled climatic conditions and in field-grown trees. Extracts were purified by immunoaffinity chromatography and high-performance liquid chromatography. Cytokinin levels were quantified with an enzyme-linked immunosorbent assay. The cytokinin content was low in buds and needles of dormant seedlings but increased during dormancy release, reaching peak values in buds just before resumption of shoot growth. Samples collected in the field also showed a marked increase in the levels of cytokinins just prior to bud burst. Further, the biological activity of applied cytokinins in activating the dormant buds of Scots pine is shown. The results indicate a probable role of cytokinins in seedlings during dormancy release.     

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.