Changes in the Endogenous Cytokinins of Bark and Buds ofSalix babylonica as a Result of Stem Girdling

Girdling of 1-year-old Salix babyionica L. plants resulted in an early accumulation of compounds which co-chromatographed with cytokinin glucosides in both the bark and buds below the girdle. In the bark the cytokinin glucosides were present in high levels in both girdled and non-girdled plants. In the buds of non-girdled plants. however, glucoside concentration was initially low but then increased rapidly after ringing and reached a maximum level prior to any visible signs of bud swell. With the onset of lateral shoot growth the glucoside cytokinins decreased while the cytokinins that co-chromatographed with zeatin and its derivatives increased. As the cytokinin glucosides are generally considered to be storage forms, their accumulation in the bark and buds below the girdle apparently does not reflect synthesis but rather transport towards a more competitive sink. In the case of Salix plants the lateral buds would appear to have the ability to hydrolyze these glucosylated zeatin derivatives and then to utilize them for bud development. It is suggested that in the presence of a functional root system lateral buds do not synthesize cytokinins de novo, but that they do have the metabolic capacity to convert cytokinins transported to them.     

Cytokinin/Auxin Control of Apical Dominance in Ipomoea nil

Although the concept of apical dominance control by the ratioof cytokinin to auxin is not new, recent experimentation withtransgenic plants has given this concept renewed attention.In the present study, it has been demonstrated that cytokinintreatments can partially reverse the inhibitory effect of auxinon lateral bud outgrowth in intact shoots of Ipomoea nil. Althoughless conclusive, this also appeared to occur in buds of isolatednodes. Auxin inhibited lateral bud outgrowth when applied eitherto the top of the stump of the decapitated shoot or directlyto the bud itself. However, the fact that cytokinin promotiveeffects on bud outgrowth are known to occur when cytokinin isapplied directly to the bud suggests different transport tissuesand/or sites of action for the two hormones. Cytokinin antagonistswere shown in some experiments to have a synergistic effectwith benzyladenine on the promotion of bud outgrowth. If theratio of cytokinin to auxin does control apical dominance, thenthe next critical question is how do these hormones interactin this correlative process? The hypothesis that shoot-derivedauxin inhibits lateral bud outgrowth indirectly by depletingcytokinin content in the shoots via inhibition of its productionin the roots was not supported in the present study which demonstratedthat the repressibility of lateral bud outgrowth by auxin treatmentsat various positions on the shoot was not correlated with proximityto the roots but rather with proximity to the buds. Resultsalso suggested that auxin in subtending mature leaves as wellas that in the shoot apex and adjacent small leaves may contributeto the apical dominance of a shoot. (Received September 24, 1996; Accepted March 16, 1997)     

The hormonal regulation of axillary bud growth in Arabidopsis

Apically derived auxin has long been known to inhibit lateral bud growth, but since it appears not to enter the bud, it has been proposed that its inhibitory effect is mediated by a second messenger. Candidates include the plant hormones ethylene, cytokinin and abscisic acid. We have developed a new assay to study this phenomenon using the model plant Arabidopsis. The assay allows study of the effects of both apical and basal hormone applications on the growth of buds on excised nodal sections. We have shown that apical auxin can inhibit the growth of small buds, but larger buds were found to have lost competence to respond. We have used the assay with nodes from wild-type and hormone-signalling mutants to test the role of ethylene, cytokinin and abscisic acid in bud inhibition by apical auxin. Our data eliminate ethylene as a second messenger for auxin-mediated bud inhibition. Similarly, abscisic acid signalling is not to be required for auxin action, although basally applied abscisic can enhance inhibition by apical auxin and apically applied abscisic acid can reduce it. By contrast, basally applied cytokinin was found to release lateral buds from inhibition by apical auxin, while apically applied cytokinin dramatically increased the duration of inhibition. These results are consistent with cytokinin acting independently to regulate bud growth, rather than as a second messenger for auxin. However, in the absence of cytokinin-signalling mutants, a role for cytokinin as a second messenger for auxin cannot be ruled out.     

The role of long range, local and direct signalling molecules during chick feather bud development involving the BMPs, follistatin and the Eph receptor tyrosine kinase Eph-A4.

The development of the feather buds during avian embryogenesis is a classic example of a spacing pattern. The regular arrangement of feather buds is achieved by a process of lateral inhibition whereby one developing feather bud prevents the formation of similar buds in the immediate vicinity. Lateral inhibition during feather formation implicates a role of long range signalling during this process. Recent work has shown that BMPs are able to enforce lateral inhibition during feather bud formation. However these results do not explain how the feather bud escapes the inhibition itself. We show that this could be achieved by the expression of the BMP antagonist, Follistatin. Furthermore we show that local application of Follistatin leads to the development of ectopic feather buds. We suggest that Follistatin locally antagonises the action of the BMPs and so permits the cellular changes associated with feather placode formation. We also provide evidence for the role of short range signalling during feather formation. We have correlated changes in cellular morphology in feather placodes with the expression of the gene Eph-A4 which encodes a receptor tyrosine kinase that requires direct cell-cell contact for activation. We show that the expression of this gene precedes cellular reorganisation required for feather bud formation.     

Hormones and Apical Dominance in the Fern Davallia

Lateral buds of the fern Davallia trichomanoides are releasedfrom inhibition by the removal of the main shoot apex. However,auxin is not capable of substituting for the apex in decapitatedshoots nor can auxin in shoot tips be detected by bioassay orextraction and chromatography. Expanding leaves of this speciescontain auxin, but these organs are not responsible for inhibitionof lateral bud growth. The response of lateral buds to an exogenouslyapplied cytokinin does not result in initial bud break. It isconcluded that the hormonal factors known to govern apical dominancein seed plants are not responsible for the regulation of differentialbud expansion in this fern.     

The apical control of lateral bud development in excised shoot tips of Cuscuta reflexa cultured in vitro

Excised shoot tips of Cuscuta reflexa Roxb. (dodder), a rootless and leafless angiospermic plant parasite, were cultured in vitro for the study of the control of lateral bud development by the apex. In a chemically defined medium lacking hormones, the basal bud alone developed into a shoot. The addition of coconut milk to the growth medium induced the activation of multiple lateral buds, but only a single bud developed further into a shoot. The decapitation of this shoot induced the development of another shoot and the process could be repeated. This showed the controlling effect of the apex in correlative control of bud development. Application of indole-3-acetic acid to the shoot tip explant delayed the development of the lateral bud. Gibberellic acid A₃ induced a marked elongation growth of the explant and reinforced apical dominance. The direct application of cytokinin to an inhibited bud relieved it from apical dominance. A basipetally decreasing concentration gradient of auxin may prevail at the nodes. Bud outgrowth is probably stimulated by cytokinin produced locally in the bud.     

ABA prevents the second cytokinin-mediated event during the induction of shoot buds in the moss Funaria hygrometrica

The induction of shoot buds in the moss Funaria hygrometrica is a classic and quantitative bioassay for cytokinin. This cytokinin-stimulated response can be inhibited by the plant hormone abscisic acid, ABA; the inhibition is concentration dependent and was proposed for use as a bioassay for ABA. This paper characterizes the ABA inhibition of the cytokinin-stimulated formation of shoot buds. Experiments transferring protonema between cytokinin and cytokinin plus ABA show that ABA does not interfere with the initial perception of cytokinin. Other experiments compare the results of transferring protonema from cytokinin to cytokinin-free medium or to medium with cytokinin plus ABA and reveal that ABA acts by blocking the cytokinin-mediated stable commitment of nascent buds. Extension of the technique of double-reciprocal plots to this whole-organism bioassay finds that ABA is not a competitive inhibitor of cytokinin. Analysis of the ABA inhibition of bud formation identifies a new regulatory step in the developmental process of bud formation in mosses.     

Cytokinin Function: Increase in [Methyl−14C] Metabolism to Phosphatidylcholine and a Decrease in Oxidation to Carbon Dioxide−14C

Axillary buds of Nicotiana tabacum L. cv. Maryland Catterton normally suppressed by apical dominance are released from dormancy with 6-benzylaminopurine. This work was done to determine the change in the [methyl^?14C] metabolism from methionine during bud stimulation with cytokinin. Dormant buds metabolize [methyl^?14C] -methionine to ¹⁴CO₂ more effectively than buds released from dormancy. This oxidation of the methyl group is inhibited with benzylaminopurine. On the other hand, the methylation of polar membrane components, including phosphatidylcholine, is enhanced by the cytokinin during the period preceding the increase in bud weight. The interpretation is presented that the enhanced synthesis of membrane components, as well as the preservation of the methyl groups, are mechanisms for the cytokinin release of bud dormancy with 6-benzylaminopurine.     

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.     

Regeneration in Streptocarpus Leaf Discs and its Regulation by Temperature and Growth Substances

The formation of adventitious buds and roots in leaf discs of Streptocarpus x bybridus‘Constant Nymph’ were both stimulated by relatively low temperatures (12 and 18°C) applied to isolated discs or to the growing plants before leaf harvest. Auxins also promoted both bud and root formation, the optimum concentration for rooting always being one to two orders of magnitude higher than the optimum for budding. Cytokinins had only a small stimulatory effect on bud formation. At higher concentrations it was inhibitory and even counteracted the stimulatory effect of auxin on bud formation. As usual, root formation was inhibited by cytokinin. GA₃ inhibited both bud and root formation but the inhibition was reversible by auxin. In presence of optimum auxin levels abscisic acid enhanced bud formation. It had little effect on root formation except for an inhibition at high concentrations. The effects of exogenous auxin and cytokinin suggest that Streptocarpus leaves have a high and non-limiting level of endogenous cytokinin with auxin as the limiting factor for both root and bud formation. This would also explain the exceptionally high regeneration ability of this plant.     

“Lateral Control”: Phytohormone Relations in the Conifer Treetop and the Short- and Long-Term Effects of Bud Excision in Abies nordmanniana

In a conifer tree, such as Nordmann fir, Abies nordmanniana Spach, the leader bud and its immediate surroundings play a decisive role in crown architecture. As subapical branch buds are segregated from the leader meristem, resource allocation between ortho- and plagiotropic growth is determined. The relationship between treetop buds in young trees was studied in the natural state and after surgical removal in early July of either the leader bud (decapitation) or the subapical whorl branch buds (destipitation). The two bud types showed consistent cytokinin profile differences but similar seasonal dynamics in cytokinins and auxin (IAA). After bud excision, ZRP increased dramatically in the subapical stem within 1 h, followed by ZR within 1 week. Supernormal levels of ZR were maintained through autumn and persisted in spring in the destipitated trees, but had returned to normal in the decapitated trees. The treetop buds remaining after bud excision experienced an immediate decrease in most cytokinins, followed, however, by a large surplus later in the season. The following spring this high level persisted in the leader bud of destipitated trees, but not in whorl buds of decapitated trees. Conspicuous growth pattern changes followed from destipitation, but few from decapitation. Growth reactions suggest that resource allocation to main branch buds inhibits leader growth in normal trees, a kind of “lateral control.” Auxin and ABA content in buds and stems was largely unaffected by treatments. Data suggest that subapical leader tissues beneath the apical bud group are a primary source of cytokinin regulation.     

Control of bud inhibition in Cyperus

Summary The axillary buds in the leaf crown of Cyperus alternifolius seedlings remain completely inhibited although the shoot is determinate and has no active apex. Buds can be released by detachment of the crown from the plant or by direct application of aqueous enzyladenine (BA), and grow out as inflorescences or vegetative shoots. These arise from activated growth centers of the primordial reproductive branch system which is enclosed within the prophyll of the inhibited bud. Buds are also released by the growth retardant, (2-chloroethyl) trimethylammonium chloride (CCC). Gibberellic acid maintains bud inhibition in detached crowns and inhibits bud release caused by CCC or BA. Naphthaleneacetic acid somewhat reduces BA-induced bud release and causes abnormal root proliferation in CCC-treated crowns. It is suggested that a high level of gibberellin within the crown, possibly in relation to a low level of cytokinin, maintains bud inhibition.     

Cytokinin induces the developmentally restricted synthesis of an extracellular protein in Physcomitrella patens

Early development of the moss Physcomitrella patens follows a simple course leading to the formation of a filamentous protonema containing only two cell-types, chloronema and caulonema. The addition of the hormone cytokinin leads to the induction of multicellular buds from such protonema. The spectrum of extracellular proteins (ECPs) synthesized by P. patens has been investigated at defined stages of development and under defined hormone treatments. It is found that in contrast to the limited changes in intracellular protein synthesis detectable, in the extracellular environment major and specific changes in the patterns of proteins synthesized occur. For example, the presence of caulonema cells is characterized by the synthesis of a 25 kDa ECP whereas early chloronema differentiation is distinguished by the presence of a 38 kDa ECP. The analysis of the pattern of ECPs synthesized by developmental mutants altered in bud formation, and in response to cytokinin in tunicamycin treated protonema (in which bud induction is blocked) indicate that the synthesis of a 14 kDa ECP is specifically induced by cytokinin. This protein represents a novel cytokinin-induced ECP. These data show that the differentiation of particular cell types in plants is associated with the synthesis of particular ECPs, and suggest that hormones which induce specific morphogenic events may do so via the synthesis of specific ECPs.     

Changes in the lanthanum distribution following decapitation of the sporophytes of an aquatic fern,Marsilea drummondii A. Br.

Summary The active terminal bud and the quiescent lateral buds and corresponding nodes inserted at different levels on the main rhizome ofMarsilea drummondii were examined with the EM afterin vivo feeding with lanthanum nitrate. These tracer experiments demonstrate that all the buds are fed by their phloem cells. In the lateral bud axis the labelling of the sieve elements apoplast indicates that a solute transfer took place in the node between xylem and phloem via xylem transfer cells. La³⁺ deposits are completely absent from the apical dome of inhibited buds indicating that the walls of the quiescent meristematic cells are not permeated by the tracer. The removal of the terminal bud has two effects. It rapidly (in 2 hours) allows the lanthanum to penetrate the lateral bud tip walls at a stage when no fine structural changes are discernable and to bind to the outer surface of the plasmalemma as it does in the active terminal bud. This study including inhibited buds and buds released from apical dominance support the view that changes in the state of the cell surface (cell wall and plasma membrane) may be a prerequisite for the resumption growth activity.This study was supported in part by a grant from the Centre National de la Recherche Scientifique to L.Sossountzov (AI 031275).     

The influence of cytokinin pulse treatments on adventitious bud formation on vegetative buds of Picea abies

Resting vegetative buds of Picea abies collected from phytotron-grown rooted cuttings of 24-year-old trees or a 12-year-old hedge were tested for their capacity to form adventitious buds after various cytokinin treatments. The most effective method for obtaining a high yield of adventitious buds within 8 weeks was to pulse treat the buds in 250 M BA for 3 h and then culture them on medium containing 5 M each of BA and kinetin for 1 week. The developmental pattern for adventitious bud production, with the formation of 10 to 20 adventitious buds per bud, was similar for all tested genotypes, although the number of buds giving rise to adventitious buds varied significantly. The capability of some clones to form adventitious buds was correlated to endogenous cytokinin content. The clone which contained most endogenous cytokinin in its resting bud had the highest potential for adventitious bud formation.     

Effect of Abscisic Acid and Other Plant Hormones on Growth of Apical and Lateral Buds of Seedlings

The effect of abscisic acid (AbA) on the growth of lateral and apical buds was studied in seedlings of Pisum sativum and some other species. The hormone was applied in three different ways: 1) directly to the lateral bud on the second node of decapitated pea seedlings as 5 μI droplets in an ethanolic solution; 2) to the cut surface of decapitated seedlings: 3) to the apical bud of intact plants. AbA directly applied in amounts of 5 to 0.1 μg to the lateral bud of the second node of decapitated seedlings had a strong inhibitory effect on the bud. Application to the cut surface of seedlings decapitated about 5 mm above the second node resulted in slight inhibition of the lateral bud on the second node and in growth promotion of the bud on the first node. When AbA at 10 to 0.1 μg was applied to the apical bud of intact seedlings, the growth of this bud was inhibited but the lateral buds grew out. It is concluded that the release of the lateral buds from apícal dominance is the result of the inhibitory effect of AbA on growth of the apical bud and of low transport of AbA. This conclusion is supported by application of GA₃ and IAA, individually and each combined with AbA.     

The effect of decapitation on the levels of IAA and ABA in the lateral buds of Betula pendula roth

The level of IAA and ABA in lateral buds of birch shoots 24 h and 5 days after the decapitation of the apical bud was determined. Twenty four hours after decapitation, when visible signs of outgrowth of lateral buds were not observed yet, an increase in the level of IAA and a decrease of ABA, as compared with the buds of non-decapitated shoots, was found. Five days later, when lateral buds were in the period of intensive outgrowth, a decrease in the levels of IAA and ABA was observed. It has been suggested that removing the source of auxin, by the decapitation of the apical bud makes possible the lateral buds to undertake the synthesis of their own auxin. It could lead to the decrease in the content of ABA. These all events could create suitable conditions for the outgrowth of lateral shoots.     

The Distribution of Hormones in Relation to Apical Dominance in Brussels Sprouts (Brassica oleracea var. gemmifera L.) Plants

The lateral buds of intact Brussels sprout plants containedless auxin and gibberellin than the main apex. When the apexwas removed the auxin content of the top lateral buds increasedwithin 2 days, but gibberellin activity did not increaseuntilshoot extension was apparent. Auxin application to the cut surfaceof decapitated plants caused lateral bud inhibition, but didnot completely prevent bud growth. Both auxin and gibberellinactivity in the plant apex decreased with increasing age, butonly gibberellin activity decreased in the lateral buds. Theauxin content of the lateral buds on intact plants increasedwith time. It is suggested that in Brussels sprouts, lateral bud inhibitionis due to sub-optimal auxin activity, and that decapitationinduces an auxin increase in these buds which then grow out.Lateral shoots are produced following decapitation of youngplants because the gibberellin content of the lateral buds isrelatively high. Only bud swelling occurs in decapitated olderplants because the gibberellin content of the buds is too lowto stimulate shoot extension. It is concluded that these results support the theory that hormone-inducednutrient diversion may control lateral bud development.