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Division of Biological Sciences, Department of Ecology and Evolutionary Biology, University of Kansas,Lawrence, Kansas 66045
Received for publication March 31, 1999. Accepted for publication July 13, 1999.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSLITERATURE CITED |
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Key Words: bud formation • cytokinin • Funaria hygrometrica • moss • phenylurea • plant developmental biology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSLITERATURE CITED |
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; Bhatla, 1994
). Indeed, the ability to count buds on protonemata has allowed bud formation to be a classic bioassay for cytokinin activity. Exposure to most cytokinins results in log-linear dose-dependent increases in bud number. Stimulation of bud formation can usually be detected at concentrations of 
10-8 mol/L, and maximal numbers of buds result from exposures to optimal concentrations of 10-6 mol/L (Brandes and Kende, 1968
; Valadon and Mummery, 1971
).
The responsiveness to cytokinin seems to be a universal property of mosses and is documented in the literature by a large number of papers that examine the effects of various substituted adenines, both natural and synthetic, on bud formation in a wide range of mosses (summarized in Chopra and Kumar, 1988
). These surveys usually find one or the other of the cytokinins is significantly more effective at inducing buds in a particular species, using either of two end-points: the absolute number of buds produced at equimolar concentrations or as the compound that stimulates bud formation at the lowest concentration. No overarching structure–activity relationships have come from this work, perhaps in part because other factors in the bioassay conditions may also be playing a role. Recently, for example, the choice of gelling agent has been shown to modulate the bud-forming response of moss protonema (Hadeler, Scholz, and Reski, 1995
).
While the natural cytokinins and the synthetic cytokinins based on them are all substituted adenines, a series of phenylureas have also been shown to have cytokinin activity (Bruce and Zwar, 1966
). These compounds were developed for commercial use as defoliants in cotton and other crops, are active in a number of cytokinin bioassays, and are now widely used as cytokinins in higher plant tissue culture and micropropagation protocols (Shudo, 1994
). Although bud formation in moss is a classic demonstration of the dramatic response of plants to cytokinin, a survey of the literature finds only incidental comments using this bioassay with the phenylurea cytokinins (no activity: Brandes and Kende, 1968
; Hahn and Bopp, 1968
; low activity: Valadon and Mummery, 1971
).
In this paper we report that the phenylurea cytokinins chloro-pyridyl-phenylurea, CPPU, and thidiazuron, TDZ, will induce bud formation in the moss Funaria hygrometrica, and this induction is dose-dependent. The related compound, diphenylurea, DPU, an active cytokinin in other bioassay systems (Shudo, 1994
), induces only very small numbers of buds in moss, and this stimulation is dose-independent from 10-9 mol/L to 10-6 mol/L. We further show that DPU does not inhibit the stimulation of bud formation by the substituted adenine cytokinin, benzyladenine, even when present in 20-fold excess.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSLITERATURE CITED |
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), are maintained by serial subculture, and are stored as cryopreserved stocks (Christianson, 1998a
).
As described previously (Christianson, 1998b
), rather than select light-grown colonies of moss just as the first caulonemal (cytokinin-responsive) filaments appear (Brandes and Kende, 1968
), experiments in our laboratory use the cytokinin-responsive protonema generated as dark-grown caulonema (Doonan et al., 1986
). Three small pieces of a stock culture are inoculated onto sterile 7-cm Whatman number 1 filter papers placed on basal medium and incubated in the dark for 14 d. Culture plates are oriented edgewise so the negatively geotropic protonemal filaments grow along the surface of the filter papers. The collection of filaments derived from each spot of inoculum is termed a "colony", and bud formation after exposure to cytokinin and continuous light (Sylvania GroLux bulbs) is quantified by counting the number of buds formed by each protonemal colony 7 d after the initial exposure to cytokinin. Since mean numbers of buds and the variance associated with those means are not independent, statistical comparisons between treatments use data transformed as 1/(x + 1) to achieve homogeneity of variance (details in Christianson and Warnick, 1983
); significance was judged at the 5% level.
The amount of innoculum and the duration of growth in the dark can be varied, resulting in colonies that make 60 buds on our BA standard medium, colonies that make 200 buds on our BA standard medium (compare Fig. 1 and Fig. 2), or colonies with other amounts of potential for bud formation. Since replicate experiments, done with colonies that differ in bud-forming potential, give identical results when means are converted to percentage of the mean of the BA standard, we are confident that our results report general properties of bud formation from moss protonema and do not depend on the absolute numbers of buds being formed, i.e., the mass or age of the protonema being assayed.
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formulation of Knop's major salts, including iron supplied as FeNaEDTA, and the microelements used by Fred Sack (Ohio State University, Columbus, Ohio): 70 µmol/L H3BO3, 14 µmol/L MnCl2, 10 µmol/L NaCl, 1 µmol/L ZnSO4, 0.5 µmol/L CuSO4, 0.2 µmol/L NaMoO4, and 10 nmol/L CoCl2. Medium was usually solidified with 8 g/L Difco BactoAgar; the increased gel strength of 18 g/L was needed for plates to be incubated edgewise in the dark. All media included sucrose at 10.2 g/L. Medium for maintenance of stock cultures included parachloroisobutyric acid at 20 µmol/L to retard the formation of caulonema (Sood and Hackenberg, 1979
). Medium for induction of buds included the cytokinin benzyladenine added before autoclaving, or was supplemented with a phenylurea cytokinin, 1,3-diphenylurea (carbanilide, DPU), N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) or 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (thidiazuron, TDZ) (SIGMA Chemical Company) from stock solutions prepared in DMSO immediately before pouring. Control experiments showed no effect of these levels of DMSO on bud formation. |
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSLITERATURE CITED |
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), and this treatment was used as a standard for comparisons of the activity of the phenylurea cytokinins. All experiments included separate no-cytokinin controls to confirm that the protonema would not form buds (by 7 d) in the absence of transfer and exposure to exogenous cytokinin. Treatment with two of the phenylurea cytokinins (CPPU and TDZ) led to maximal stimulation of bud formation (means at best concentrations, not significantly different from mean at 10-6 mol/L BA), while treatment with the remaining compound (DPU) led only to a very small stimulation of bud formation (6.7% of the BA standard, significantly different from the BA standard; also significantly different from the no-cytokinin control, P < 0.01) (Fig. 1).
Treatments with the active phenylureas, like treatments with substituted adenine cytokinins, result in concentration-dependent stimulation of buds. For CPPU, this dose-dependent increase extends over a larger concentration range (10-9 through 10-6 mol/L than we see for BA (from 10-7 mol/L to 10-6 mol/L) (Fig. 1). As reported for substituted adenine cytokinins (including BA on our Funaria isolate, data not shown) (Brandes and Kende, 1968
; Hahn and Bopp, 1968
; Valadon and Mummery, 1971
), treatment with supra-optimal levels of the phenylurea cytokinins results in less than maximal numbers of buds or even toxicity. At 10-5 mol/L, CPPU treatment results in no buds. At the highest level tested (3 x 10 - 5 mol/L), protonemata appear brown and show no evidence of any growth during the 7 d of CPPU exposure; when these protonemata are transferred to maintenance medium (no CPPU), only one of nine colonies tested was able to regrow, and that colony regenerated from a single surviving segment of one protonemal filament.
In contrast to the broad dose-response curve obtained with CPPU, TDZ shows a log-linear dose-response curve similar to the curves described for BA and other substituted adenines, going from essentially no stimulation of buds to maximal stimulation of buds over an 30-fold concentration range (Fig. 1). TDZ is active, however, over a slightly higher range of concentrations than is BA. This difference might reflect differences in affinity for hormone receptors, but it could also reflect differences in hormone uptake or metabolism.
Since some phenylureas are extraordinarily active in higher plant bioassays (to 10-13 mol/L; Shudo, 1994
), the small stimulation of bud formation by DPU at micromolar concentrations might be the effect of supra-optimal but nontoxic concentrations of this hormone. Bioassay of picomolar concentrations, however, resulted in even fewer buds than the small stimulations of bud formation seen from micromolar treatments. Statistical examination of these data does not find a significant dose-dependent relationship between concentration of DPU and the small numbers of buds produced by treatments from 10-9 to 10-6 mol/L. This confirms what casual inspection of these data suggests: treatments with DPU result in the same stimulation of bud formation, 10 buds per colony, over a 1000-fold concentration range (Fig. 1). Valadon and Mummery (1971) did report that DPU was able to induce buds in Funaria at concentrations from 4.7 x 10-8 to 4.7 x 10-5 mol/L and that this activity was maximal at 4.7 x 10-7 mol/L. At that concentration, treatment with DPU resulted in 6.3% the number of buds that resulted from treatment with the optimal level of benzyladenine, a finding entirely consistent with our results.
It is typical for biologically active chemicals such as herbicides or antibiotics to reach maximal effects within a 30-fold concentration range (Hartley and Graham-Bryce, 1980
). The lack of a strong dose-dependent relationship for the stimulation of bud formation by DPU as well as the fact that DPU produces this minimal stimulation at concentrations spanning three orders of magnitude are both highly unusual features of the interaction of DPU and the protonema of moss.
BA combined with DPU
This combination of minimal activity for DPU (7% the BA standard) plus a three-order-of-magnitude concentration range is difficult to reconcile with the usual biochemical descriptions of how compounds affect cells. If, for example, cells do not take up DPU from the medium very efficiently (or metabolize it quickly to an inactive form), DPU might not stimulate bud formation as well as some other cytokinin. But under this premise, higher concentrations of DPU should give more stimulation than lower concentrations, something we do not see. It would be possible to explain the flat dose-response curve by proposing that DPU will bind to the cytokinin receptors in the cell, but unlike the binding of other cytokinins to these receptors, the binding of DPU neither activates the receptor nor allows the ligand to be released from the receptor. Relatively low concentrations of DPU would bind all the available receptor sites, and increased concentrations of DPU would lead to the same low level of stimulation of bud formation as exposure to the lower concentration. Such effects on enzymatic activity are well known (Dixon and Webb, 1979
). Other premises about how DPU might bind, activate, and be released from the cytokinin receptors in moss cells are possible (Dixon and Webb, 1979
), of course. All these premises predict that DPU will complete in some way with active cytokinins for the cytokinin receptors, and this is a prediction that can be tested.
Recent progress in identifying probable cytokinin receptors or response elements in higher plants (Kakimoto, 1996
) might someday allow a direct test of such premises, measuring binding, activation, and release of DPU and other cytokinins. Fortunately, it is also possible to test these premises by simple bioassay experiments. If DPU binds ineffectively on cytokinin receptors, it will compete with BA, presumably bound and released in cycles at the same sites on the cytokinin receptors. This competition will be most effective at low concentrations of BA. Indeed, when inactive phenylureas and BA were bioassayed in mixtures using the tobacco callus bioassay system, the phenylureas were shown to inhibit the action of BA; analysis via classic Lineweaver-Burk plots indicated that the substituted adenine cytokinins and the phenylureas compete for the same binding site on the cytokinin receptor (Shudo, 1994
).
Our experiments measured bud formation from moss protonema exposed to mixtures of DPU and BA. We compared the numbers of buds produced by protonema exposed to increasing concentrations of cytokinin, in the absence and in the presence of DPU, and find no evidence for competition between BA and DPU in moss (Fig. 2). The addition of 1 or 2 µmol/L DPU did not alter the standard log-linear increase in numbers of buds from 0.2 to 2 µmol/L BA. While sound biochemical principles predict that DPU and BA will compete for receptors (Dixon and Webb, 1979
), and although such competition has been demonstrated with tobacco callus (Shudo, 1994
), experiments using bud formation in moss find no evidence for any type of formal kinetic interaction, competitive, uncompetitive, or noncompetitive, between BA and DPU.
Although the phenylurea cytokinins are usually thought to act directly as cytokinins, there is at least some evidence in some species for an indirect mechanism of action. In those cases, the phenylureas have been shown to affect cytokinin metabolism in treated tissues (Hare and Van Staden, 1994
). Despite the availability of hormone-insensitive mutants (reviewed in Bhatla, 1994
), the biochemical knowledge of hormone metabolism in mosses is still too poorly known to permit fine discrimination between direct and indirect mechanisms of action in moss (but see Sztein et al. [1995]
for auxin metabolism). We do know that bud formation in mosses involves not one but two distinct interactions with cytokinin (Brandes and Kende, 1968
; Christianson, 1998b
). If both the initial perception of cytokinin and the cytokinin-mediated commitment of nascent buds use the same cytokinin receptor, DPU and BA would be expected to compete for receptors as described above, and the lack of formal interaction we report in this paper might mean that DPU (and by extension, the other phenylurea cytokinins) acts by an indirect mechanism in moss.
Of the two temporally distinct cytokinin requirements for bud formation, it is the second event that is responsible for the dose-dependency of bud number (Christianson, 1998b
). Preliminary experiments, assaying for the ability of DPU to initiate bud formation, find that DPU, like BA, can and does activate the cytokinin receptor controlling the initial responses. If DPU has little or no affinity for the receptor triggering the second event, DPU treatments will produce few to no buds (as we observe), and kinetic analysis using bud number would find no evidence for competition with benzyladenine (as we observe). The experiments with phenylurea cytokinins reported in this paper raise the intriguing possibility that bud induction in mosses involves two chemically distinct cytokinin receptors.
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSLITERATURE CITED |
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Bopp, M. 1990 Hormones of the moss protonema. In R. N. Chopra and S. C. Bhatla [eds.], Bryophyte development: physiology and biochemistry, 55-77. CRC Press, Boca Raton, FL.
Brandes, H., and H. Kende. 1968 Studies on cytokinin-controlled bud formation in moss protonema. Plant Physiology 43: 827–837.
Bruce, M. I., and J. A. Zwar. 1966 Cytokinin activity of some substituted ureas and thioureas. Proceedings Royal Society. London, Series B, 165: 245–265.
Christianson, M. L. 1998a A simple protocol for cryopreservation of mosses. Bryologist 101: 32–35.[ISI]
———. 1998b The quantitative response to cytokinin in the moss Funaria hygrometrica does not reflect differential sensitivity of initial target cells. American Journal of Botany 85: 144–148.[Abstract]
———, and D. A. Warnick. 1983 Competence and determination in the process of in vitro shoot organogenesis. Developmental Biology 95: 288–293.[CrossRef][ISI][Medline]
Chopra, R. N., and P. K. Kumar. 1988 Biology of bryophytes. John Wiley&Sons, New York, NY.
Dixon, M., and E. C. Webb. 1979 Enzymes, 3rd ed. Academic Press, New York, NY.
Doonan, J. H., G. I. Jenkins, D. J. Cove, and C. W. Lloyd. 1986 Microtubules connect the migrating nucleus to the prospective division site during side branch formation in the moss, Physcomitrella patens. European Journal of Cell Biology 41: 157–164.[ISI]
Hadeler, B., S. Scholz, and R. Reski. 1995 Gelrite and agar differently influence cytokinin–sensitivity of a moss. Journal of Plant Physiology 146: 369–371.[ISI]
Hahn, H., and M. Bopp. 1968 A cytokinin test with high specificity. Planta 83: 115–118.[CrossRef][ISI]
Hare, P. D. and J. Van Staden. 1994 Inhibitory effect of thidiazuron on the activity of cytokinin oxidase isolated from soybean callus. Plant and Cell Physiology 55: 1121–1125.
Hartley, G. S., and I. J. Graham-Bryce. 1980 Physical principles of pesticide behaviour. Academic Press, London, UK.
Kakimoto, T. 1996 CKI1, a histidine kinase homologue implicated in cytokinin signal transductioon. Science 274: 982–985.
Saunders, M. J. and P. K. Hepler. 1983 Calcium antagonists and calmodulin inhibitors block cytokinin-induced bud formation in Funaria. Developmental Biology 99: 41–49.[CrossRef][ISI][Medline]
Shaw, A. J. 1991 The genetic structure of sporophytic and gametophytic populations of the moss, Funaria hygrometrica. 45: 1260–1274.
Shudo, K. 1994 Chemistry of phenylurea cytokinins. In D. W. S. Mok and M. C. Mok [eds.], Cytokinins: chemistry, activity and function, 35–42. CRC Press, Boca Raton, FL.
Sood, S., and D. Hackenberg. 1979 Interaction of auxin, antiauxin and cytokinin in relation to the formation of buds in moss protonema. Zeitschrift für Pflanzenphysiologie 91: 385–397.
Sztein, A. E., J. D. Cohen, J. P. Slovin, and T. J. Cooke. 1995 Auxin metabolism in representative land plants. American Journal of Botany 82: 1514–1521.[CrossRef][ISI]
Valadon, L. R. G., and R. S. Mummery. 1971 Quantitative relationship between various growth substances and bud production in Funaria hygrometrica: a bioassay for abscisic acid. Physiologia Plantarum 24: 232–234.[CrossRef]
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