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.     

Phenylurea cytokinins assayed for induction of shoot buds in the moss Funaria hygrometrica

The induction of shoot buds from the filamentous protonema of moss is a classic bioassay for cytokinin. While a large literature documents this response in many species of moss and for a wide range of natural and synthetic cytokinins, to date only substituted adenine cytokinins have been examined in detail. This paper shows that at least some of the novel phenylurea cytokinins will induce bud formation in mosses. Funaria responds to thidiazuron much as it responds to benzyladenine. Exposure to either substance results in log-linear dose-dependent increases in bud number that reach similar maximal numbers of buds at the optimal concentration of compound. The related compound chloro-pyridyl-phenylurea (CPPU) is slightly less active, but induces buds over a wider range of concentration. Carbanilide (diphenylurea or DPU), an active cytokinin in other systems, induces very few buds in Funaria, but does so over a wide range of concentration. Bioassay of mixtures of benzyladenine and DPU finds no evidence of competition for cytokinin receptors. That result could support suggestions that the phenylurea cytokinins act indirectly, by altering endogenous cytokinin metabolism, but we favor another interpretation. Unlike other cytokinin-responsive systems, the induction of buds from moss protonema involves two cytokinin-mediated events. The number of buds is controlled by the second cytokinin-mediated event. If DPU has little or no affinity for the receptor triggering this second event, DPU treatments will produce few to no buds, and kinetic analysis using bud number would find no evidence for competition with benzyladenine. Our results would support the hypothesis that bud induction in Funaria involves two chemically distinct cytokinin receptors.     

The Effect of Ageing on Bud Differentiation in the Moss, Physcomitrium sphaericum

In the moss Physcomitrium sphaericum, we examined the numberof buds per filament, the position of buds, and the ratio ofbud-differentiated filaments when treated with cytokinin, inrelation to the increase in the number of cells per filament. When filaments of a young protonema were treated with cytokinin,many filaments did not differentiate buds. As the number ofcells in a filament increased, both the mean number of budsper filament and the ratio of bud-differentiated filaments increased.However, the position of bud differentiation was unaffectedby application of cytokinin. A higher concentration of cytokininincreased the mean number of buds per filament and the ratioof bud-differentiated filaments. The relationship between cytokinin, ageing of filaments andthe ability to differentiate buds is discussed. (Received June 17, 1985; Accepted September 9, 1985)     

Dose-Dependent Effect of Salicylates in a Moss, Funaria hygrometrica

Plant growth regulators now include more than the classic examples auxin, cytokinin, ethylene, and gibberellin, but little is known about the activity of these additional classes of molecules in nonvascular plants. The formation of buds by protonema of the moss Funaria hygrometrica is perhaps the best known and most fully characterized developmental system in the nonvascular plants. Examination of the effects of exogenously supplied salicylic acid and acetylsalicylate on this bioassay system shows that salicylates can regulate growth and development in mosses, producing a dose-dependent inhibition of bud formation. Other experiments show that this action is distinct from any direct effect on the well-known cytokinin stimulation of bud formation, occurs late in the process of bud formation, occurs prior to the stable commitment of nascent buds, and is not an effect on the outgrowth of young shoots. Because mosses are the sister clade to the vascular plants, these results suggest that the ability to perceive and transduce salicylate signals is an ancient feature of land plant physiology.     

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.     

Localization of cytokinin responses in the mossFunaria hygrometrica

In the protonema ofFunaria hygrometrica, predetermined cells run through a period of sensitivity to cytokinins, as part of the morphogenetic system with respect to bud formation. The cytokinin sensitivity is highest immediately after the formation of cells in question. The cells can react if enough hormone is present during the sensitive phase. Kinetin is cleaved very rapidly and at an increasing rate. Most probably the concerned enzyme (“kinetin oxidase”) is induced by kinetin. Therefore, cells formed after the beginning of the treatment do not respond by forming buds.     

Development of flower buds in thin-layer cultures of floral stalk tissue from tobacco: Role of hormones in different stages

The in vitro development of flower buds was studied on tissue explants of epidermis and subepidermal cortex from the flower stalks of Nicotiana tabacum L. cv. Samsun. The number of flower buds formed depended mainly on cytokinin concentration. Auxin acted as a modifier in a complex way. In early development, NAA at 1 μ M decreased the number of buds initiated and delayed bud emergence. At a later stage, auxin promoted bud outgrowth at the same concentration. Optimal results were obtained when explants were first incubated at low auxin concentration for 3–5 days and subsequently transferred to an elevated auxin level. Physiological processes that lead to flower bud initiation start very soon after the onset of incubation. This was inferred from experiments whereby explants were first cultured at an inductive cytokinin concentration and then transferred to a non-inductive hormone level.     

Relations between cytokinin level, bud development and apical control in Norway spruce, Picea abies

In conifers such as Norway spruce, the extent of shoot growth is predetermined by the size and number of embryonal organs of the buds laid down the previous year. As it is known that cytokinins have a key role in bud development a possible hypothesis is that the level of cytokinin in the buds during their formation determines their size and complexity. As a first step to test this hypothesis we compared cytokinin levels in buds of different size of annual shoots from 15- to 20-year-old trees of Picea abies (L.) Karst. Apical buds from the leaders, and from branches in lower parts of the trees, were collected in April, July and August. The difference in size of the buds and the shoots growing from them was considerable in these three positions. Extracts were purified by immunoaffinity columns, and the retained compounds were separated by high-performance liquid chromatography (HPLC). Quantification was made by enzyme-linked immunosorbent assay (ELISA), and the accuracy of this method was checked by measurements with liquid chromatography-mass spectrometry (LC-MS) and UV absorption. Zeatin riboside (ZR) was the most abundant cytokinin, but isopentenyladenosine (iPA) was also present in all samples. The large apical bud of the leader contained much higher cytokinin concentrations than the considerably smaller buds from lower positions, and during the period of secondary growth in July, similar relationships were found for annual stem tissue from different positions. The possible role of ZR as a controlling factor in bud development and apical control is discussed. Our conclusion is that the level of zeatin-type cytokinins appears to play an important role in the establishment of differences in bud size and, thereby, the architecture of the tree crown.     

A cytokinin test with high specificity

Summary A cytokinin bioassay based on bud formation in 10-cell-long caulonema filaments of Funaria hygrometrica is described. The test has high specificity and sensitivity; is completed in 2 days; exhibits linearity between cytokinin concentration in the medium and bud number; and no buds are formed in the absence of a cytokinin.     

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.     

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.     

Effect of Sucrose on Starch Accumulation in and Adventitious Bud Formation on Embryos of Picea abies

Adventitious buds were initiated on embryos of Picea abies (L.)Karst. after a pulse treatment with cytokinin. The initial stagesof bud formation could take place on culture medium lackingsucrose, but sucrose was required for further development ofmeristematic centres into bud primordia and buds. Sucrose atone per cent was optimal for adventitious bud formation. Embryoscultured on media containing sucrose started to accumulate starchduring the first day. Starch accumulation occurred especiallyin the cortex cells where starch grains were frequently presentin the chloroplasts. The starch accumulation increased withhigher sucrose concentrations in the culture medium. Embryoscultured on medium lacking sucrose did not accumulate starchbefore the formation of meristematic centres. Starch accumulationwas never observed in meristematic cells from which adventitiousbud primordia developed. Picea abies (L.) Karst., Norway spruce, adventitious bud, starch accumulation, sucrose concentration     

Induction of flower bud formation in vitro by dihydrozeatin

Flower stalk explants of tobacco cultured on a medium with an auxin and cytokinin regenerate flower buds within 14 days. The optimal medium concentrations of dihydrozeatin (DHZ) and benzyladenine (BA) were both 1 μM. The presence of DHZ in the culture medium was only essential during an initiation period of 7 days, whereas BA was needed only during the first 4 days. The difference in length of the initiation period is neither explained by the unequal uptake rates of the cytokinins nor by differences in their conjugation. At the medium concentration optimal for bud formation, the internal concentration of DHZ was two to three times the internal concentration of BA, which could be attributed to faster uptake of DHZ. It is concluded from the combined data that DHZ is less active in inducing flower bud formation than BA and that the exogenous cytokinins play only a role during the initiation phase of bud regeneration.     

In vitro flower bud formation in tobacco: interaction of hormones

External application of auxin and cytokinin is required for the formation of flower buds on thin-layer tissue explants of Nicotiana tabacum cv Samsun. Interaction between both plant growth regulators during this regenerative process has been demonstrated with respect to speed of flower bud initiation and the number of flower buds formed. Separation in time of the hormone application during culture revealed that the cytokinin benzyladenine plays a key role in flower bud initiation whereas auxin (indoleacetic acid) stimulates in particular the differentiation of flower buds. The uptake of each hormone was proportional to the concentration supplied in the medium, and the uptake of either hormone appeared independently of the presence of the other. Metabolism studies showed the conversion of indoleacetic acid by the tissue to at least 13 metabolites after 24 h of culture. In addition, indoleacetic acid metabolism was demonstrated not to be influenced by the uptake and metabolism of benzyladenine. Taken together the results indicate that the interaction of auxin and cytokinin with respect to in vitro flower bud formation is indirect, i.e. does not take place at the level of hormone uptake or metabolism but at some step in the cascade of processes they initiate.     

The accumulation and metabolism of plant growth regulators during organogenesis in cultures of thin cell layers of Nicotiana tabacum

The accumulation and metabolism of exogenously applied and endogenously produced auxins and cytokinins were studied in relation to organogenesis in thin cell layers of Nicotiana tabacum L. It was shown that, in order to obtain maximal flower bud formation, both exogenous auxin and cytokinin needed to be present during the first 4 days of culture (to the formation of a subepidermal meristematic zone) whereas cytokinins needed to be present for at least 4 days more (until formation of organogenic centres). Explants taken from floral branches have higher endogenous indole-3-acetic acid (IAA) levels compared with explants from the basal part of the stem which form only vegetative buds. This might be related to a different IAA metabolism in these two types of explants as was shown by the different accumulation of exogenously applied IAA. Both 'floral' and 'vegetative' cells layers contained comparable amounts of zeatin riboside (ZR) as their major cytokinin. Free bases, zeatin (Z) and dihydrozeatin [(diH)Z], given exogenously, were largely metabolised to their respective ribosides. The observation that Z was less effective than (diH)Z in the induction of flower buds could be related to (diH)ZR apparently not being a substrate for cytokinin oxidase.     

Induction of flower bud formation in vitro by dihydrozeatin

Flower stalk explants of tobacco cultured on a medium with an auxin and cytokinin regenerate flower buds within 14 days. The optimal medium concentrations of dihydrozeatin (DHZ) and benzyladenine (BA) were both 1 M. The presence of DHZ in the culture medium was only essential during an initiation period of 7 days, whereas BA was needed only during the first 4 days. The difference in length of the initiation period is neither explained by the unequal uptake rates of the cytokinins nor by differences in their conjugation. At the medium concentration optimal for bud formation, the internal concentration of DHZ was two to three times the internal concentration of BA, which could be attributed to faster uptake of DHZ. It is concluded from the combined data that DHZ is less active in inducing flower bud formation than BA and that the exogenous cytokinins play only a role during the initiation phase of bud regeneration.     

Cytokinin activation and redistribution of plasma-membrane ion channels in Funaria

I have investigated changes in electrical current across the plasma membrane that occur during cytokinin-induced bud formation in Funaria hygrometrica Hedw., using a non-intrusive vibrating microelectrode. Before cytokinin treatment the target caulonema cells have maximal inward current at the nuclear region. After cytokinin treatment inward current increases twofold along the length of the cell. Within minutes, however, current decreases at both the nuclear zone and the proximal end while increasing at the distal end of target cells, preceding and predicting the presumptive division site. Inward current at the distal end falls to resting levels after establishment of a bulging growth zone, and remains low around developing buds. This current is blocked by gadolinium nitrate, a Ca²⁺-uptake inhibitor, indicating a Ca²⁺ component of the current. The polarity of the target cells can be disrupted by microfilament inhibitors and cytokinin-induced buds form over the nucleus, halfway along the length of the cell. I suggest that cytokinin activates plasma-membrane ion channels which are subsequently redistributed to the distal ends of target cells by a microfilament-dependent process. Cytokinin-induced concentration of ion channels over presumptive bud sites may be envisioned to exert spatial control of cytoplasmic ion concentrations and stimulate bud formation by establishing a new growth zone, directing nuclear migration, and stimulating cell division.Abbreviations BA 6-benzyladenine - [Ca²⁺]_i intracellular calcium-ion concentration     

Micromorphological studies of adventitious bud formation on Picea abies embryos treated with cytokinin

Embryos of Picea abies (L.) Karst were pulse-treated with water or cytokinin for 2 h and then cultured on medium lacking cytokinin. Adventitious buds developed on cytokinin-treated embryos, but not on water-treated embryos. The general appearance and the surface morphology were similar on water and BA (benzyladenine)-treated embryos after 3 days. The epidermal cells were elongating after 6 days on water-treated embryos, while they were dividing on cytokinin-treated embryos. Furthermore, the cells surrounding the stomata had started to proliferate on BA-treated embryos. This was the first micromorphological sign of bud initiation. During the second week prominent meristemoids developed from these cells. A stoma was observed on the top of each meristemoid. The variation in developmental pattern of meristemoids among different embryos as well as within each embryo was small. However, during the subsequent development of bud primordia and buds, the morphological variation was significant. The meristemoids continued to develop into cone-shaped bud primordia, which successively changed shape during the transition to adventitious buds. The epidermal cells divided and the epidermis did not rupture during the formation of adventitious bud primordia. The epidermis was identified as the protoderm of the bud primordium.