phenylurea cytokinins assayed for induction of shoot buds in the ...

American Journal of Botany 86(11): 1645–1648. 1999.
BRIEF COMMUNICATION
PHENYLUREA CYTOKININS ASSAYED FOR INDUCTION OF
SHOOT BUDS IN THE MOSS FUNARIA HYGROMETRICA 1
MICHAEL L. CHRISTIANSON 2 AND JASON S. HORNBUCKLE
Division of Biological Sciences, Department of Ecology and Evolutionary Biology, University of Kansas,
Lawrence, Kansas 66045
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.
Key words: bud formation; cytokinin; Funaria hygrometrica; moss; phenylurea; plant developmental biology.
Mosses grown in vitro progress through the same developmental
stages as mosses growing in nature. In Funaria
hygrometrica, spores germinate and give rise to the
chloroplast-rich chloronema; with time, caulonemal filaments
appear, and eventually cells in these filaments begin
to undergo a special set of divisions to produce shoot
buds. These buds grow into the leafy shoots or gametophores.
Under appropriate conditions, gametangia are
produced, and sporophytes appear after successful fertilizations.
Exogenous application of plant hormones will
facilitate these transitions: auxin for the transition from
chloronema to caulonema, cytokinin to induce the formation
of buds (Bopp, 1990; 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 dosedependent
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 univer-
1 Manuscript received 31 March 1999; revision accepted 13 July
1999.
The authors thank Dr. Richard Amasino (U. Wisconsin) for asking
about the activity of phenylurea cytokinins in mosses, Dr. Richard
Schowen (U. Kansas) for enlightening discussions about analyzing kinetic
interactions, and Sharon Hagen for final preparation of the figures.
Work was supported by undergraduate research funds from the Division
of Biological Sciences, the University of Kansas General Research
Fund, NSF grant OSR-9550487, and matching support from the State
of Kansas.
2 Author for correspondence.
1645
sal 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 hygro-

1646 AMERICAN JOURNAL OF BOTANY
[Vol. 86
Fig. 1. Numbers of buds of Funaria hygrometrica induced by exposure
to standard and phenylurea cytokinins. Dark-grown protonemata
were transferred to medium supplemented with various concentrations
of benzyladenine (solid circles) or one of three phenylurea cytokinins:
thidiazuron (open circles), CPPU (open squares), or DPU (solid triangles)
and cultured in the light. Buds were counted 7 d after movement
to cytokinin-supplemented medium. N 9; error bars represent SEM.
metrica, 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.
MATERIALS AND METHODS
Plant material—The isolates of Funaria hygrometrica used in these
experiments were the ‘‘Duke’’ clone originally isolated and described
by Dr. Jon Shaw (1991) and the ‘‘Stream’’ clone, collected in northern
Alberta by MLC. Aseptic cultures were established from spores (Saunders
and Hepler, 1983), 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 (cytokininresponsive)
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
Fig. 2. The addition of diphenylurea does not alter the dose-dependent
formation of buds of Funaria hygrometrica from benzyladenine
exposure. Dark-grown protonema were transferred to medium supplemented
with various concentrations of benzyladenine and DPU at zero
(solid line), 1 mol/L (dotted line), or 2 mol/L (dashed line). Statistical
analysis finds no significant effect of DPU on the concentration-dependent
increase in bud number from BA treatment. Buds were counted 7
d after movement to cytokinin-supplemented medium. N 9; error bars
represent SEM.
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.
Culture media—Basal medium consisted of Brandes and Kende’s
(1968) formulation of Knop’s major salts, including iron supplied as
FeNaEDTA, and the microelements used by Fred Sack (Ohio State University,
Columbus, Ohio): 70 mol/L H 3BO 3,14mol/L MnCl 2,10
mol/L NaCl, 1 mol/L ZnSO 4, 0.5 mol/L CuSO 4, 0.2 mol/L
NaMoO 4, and 10 nmol/L CoCl 2. 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 mol/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

November 1999] CHRISTIANSON AND HORNBUCKLE—PHENYLUREA CYTOKININS AND MOSS
1647
Company) from stock solutions prepared in DMSO immediately before
pouring. Control experiments showed no effect of these levels of DMSO
on bud formation.
RESULTS
Phenylurea cytokinins can stimulate bud formation
in Funaria—Previous experiments established that exposure
to 10 6 mol/L benzyladenine results in near-maximal
numbers of buds from responsive protonema of Funaria
(Christianson, 1998b), and this treatment was used
as a standard for comparisons of the activity of the phenylurea
cytokinins. All experiments included separate nocytokinin
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 dosedependent
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 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 10 8 to 4.7
10 5 mol/L and that this activity was maximal at 4.7
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 indi-

1648 AMERICAN JOURNAL OF BOTANY
[Vol. 86
cated 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 mol/L DPU did not alter
the standard log-linear increase in numbers of buds from
0.2 to 2 mol/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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