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Plant Physiol, February 2002, Vol. 128, pp. 354-362
UPDATE ON CYTOKININS
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INTRODUCTION |
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 TOP
 INTRODUCTION
 BIOSYNTHESIS AND METABOLISM
BIOSYNTHESIS
METABOLISM
DEGRADATION
TRANSPORT
PERCEPTION AND SIGNAL...
CONCLUSION
LITERATURE CITED
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Cytokinins were discovered in the
search for factors that promoted division of plant cells in culture.
Naturally occurring cytokinins are
N6-substituted adenine derivatives that
generally contain an isoprenoid derivative side chain. These hormones
influence numerous aspects of plant development and physiology,
including seed germination, de-etiolation, chloroplast differentiation,
apical dominance, plant-pathogen interactions, flower and fruit
development, and leaf senescence. These processes are also influenced
by various other stimuli (e.g. light and other phytohormones), and the
physiological and developmental outcomes reflect a highly integrated
response to these multiple stimuli. For example, the classical reports of Skoog and Miller (1957)
revealed that undifferentiated callus cultures would form into roots or shoots depending on the
relative amount of cytokinins and auxin in the medium; the ratio rather than the absolute amount of these two hormones is critical. A balanced
ratio keeps the cells in an undifferentiated state, while high
cytokinin to auxin ratios promote shoot and low ratios promote root development.
In the last decade, genetic and molecular analysis of mutants has provided valuable insights into the molecular mechanisms underlying the action of other phytohormones. Cytokinin has lagged behind in such studies, but several exciting recent reports show that this is now changing. This review focuses on the dramatic recent progress in understanding cytokinin metabolism, perception, and signaling. The analysis of all three aspects is required to understand the different effects of cytokinin on plant physiology and its role as a developmental signal.
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BIOSYNTHESIS AND METABOLISM |
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TOP
INTRODUCTION
BIOSYNTHESIS AND METABOLISM
BIOSYNTHESIS
METABOLISM
DEGRADATION
TRANSPORT
PERCEPTION AND SIGNAL...
CONCLUSION
LITERATURE CITED
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Cytokinins occur as a bound form in the tRNA of most organisms, including plants, but plants also possess significant amounts of free cytokinins. Isoprenoid-type cytokinins are the most abundant, but several plant species contain adenine derivatives with aromatic substituents. In addition, there are synthetic cytokinins derived from diphenylurea (DPU) that are structurally unrelated to the adenine-type cytokinins.
The structure and conformation of the side chain are critical to the activity of the respective cytokinin. For example, one of the most abundant cytokinins in higher plants, trans-zeatin, displays a high cytokinin activity in bioassays, but the cis-isomer possesses a significantly lower activity.
In general, a given plant tissue contains several types of cytokinins and their modified forms. The distribution of the various cytokinins differs significantly between plant species. The phenotypic analysis of biosynthetic mutants could reveal the roles of specific cytokinin forms, but unfortunately neither general nor specific mutants with reduced cytokinin content have been isolated, and the overexpression of biosynthetic genes generally affects the whole pool of endogenous cytokinins. Furthermore, it is difficult to quantify cytokinins in vivo, especially with respect to their distribution within different plant tissues or organs. Our knowledge about the in vivo role of individual cytokinins and their modified forms is therefore very limited.
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BIOSYNTHESIS |
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TOP
INTRODUCTION
BIOSYNTHESIS AND METABOLISM
BIOSYNTHESIS
METABOLISM
DEGRADATION
TRANSPORT
PERCEPTION AND SIGNAL...
CONCLUSION
LITERATURE CITED
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The breakdown of tRNA was originally suggested as a possible
mechanism for cytokinin synthesis. The released cis-zeatin could subsequently be converted to active trans-zeatin by the enzyme cis-trans-isomerase (Mok and Mok, 2001). However, the slow turnover rate of tRNA is not sufficient to account for the amount of cytokinins present in plants.
Enzymatic activity that converts AMP and
dimethylallyl-diphosphate (DMAPP) to the active
cytokinin isopentenyladenosine-5'-monophosphate (iPMP) was firstly
identified in Dictyostelium discoideum (Taya et al.,
1978). Subsequently, the tmr gene (later designated as ipt) from Agrobacterium tumefaciens, which
results in root-like tumors when mutated, was shown to encode an enzyme
with similar activity (Akiyoshi et al., 1984). ipt genes
have also been identified in several other bacteria, and IPT activity
was detected in crude extracts from a variety of plant tissues,
but the plant enzymes were not purified and the corresponding genes
were not cloned.
The completion of the Arabidopsis genomic sequence enabled Takei et al.
(2001a) and Kakimoto (2001a) in independent studies to identify a total
of nine ipt-homologs, designated as AtIPT1 to
AtIPT9, by an in silico analysis. A phylogenetic analysis
suggested that AtIPT2 and AtIPT9 encode a
putative tRNA-ipt while the other seven AtIPTs formed a
distinct clade more closely related to the bacterial ipt/tmr
gene. The expression of these seven genes in Escherichia
coli resulted in the secretion of the cytokinins iP and zeatin,
confirming that they encode cytokinin biosynthetic genes (Takei et al.,
2001a). Additionally, calli overexpressing AtIPT4 under the
control of the Cauliflower mosaic virus (CaMV) 35S
promoter showed shoot regeneration even in the absence of cytokinin,
while CaMV 35S::AtIPT2 calli were still dependent
on cytokinin (Kakimoto, 2001a). Surprisingly, unlike the bacterial IPT
enzymes, purified AtIPT4 utilized ATP and ADP preferentially over AMP
as a substrate (Kakimoto, 2001). The product of the plant enzyme is
likely to be isopentenyladenosine-5'-triphosphate (iPTP) and
isopentenyladenosine-5'-diphosphate (iPDP), which can be
subsequently interconverted to zeatin (Fig.
1). Several of the AtIPT genes display distinct, tissue-specific patterns of expression, perhaps providing new insights into the sites of cytokinin production (Kakimoto, 2001b).
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Another study in Arabidopsis indicates that an alternative
cytokinin biosynthesis pathway exists in plants (Åstot et al., 2000).
The authors compared the biosynthetic rate of zeatin
riboside-5'-monophosphate (ZMP) and iPMP in wild type and transgenic
plants designed to inducibly overexpress the bacterial ipt
gene. iPMP is the direct product of the transfer of DMAPP to
AMP, and it can be converted to ZMP by an endogenous hydroxylase
activity. In vivo deuterium labeling revealed a 66-fold higher
biosynthetic rate of ZMP than that of iPMP, the product of IPT. By a
feeding experiment with two tracers, which allowed the simultaneous
determination of iPMP-hydroxylase activity and the de novo synthesis of
ZMP, it was shown that the major precursor for ZMP was not cytoplasmic
iPMP. The authors suggested the presence of an iPMP-independent
pathway, in which ZMP is directly synthesized by IPT from AMP and a yet
unidentified side chain precursor. This pathway could be strongly
inhibited by mevastatin, indicating a terpenoid origin of the
hypothetical precursor.
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METABOLISM |
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TOP
INTRODUCTION
BIOSYNTHESIS AND METABOLISM
BIOSYNTHESIS
METABOLISM
DEGRADATION
TRANSPORT
PERCEPTION AND SIGNAL...
CONCLUSION
LITERATURE CITED
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Almost all cytokinins are present in plants as both the free base
and the corresponding nucleosides and nucleotides. The interconversions among these forms are likely to be performed by enzymes involved in the
general purine metabolism, but their biological significance is unclear
(Mok and Mok, 2001). The adenine ring system can be glucosylated at the
N3-, N7-, and
N9-position. Glucosyl-conjugates at the
N7- and N9- but
not at the N3-position are usually inactive
in bioassays. It is assumed that the N7-
and N9- modifications irreversibly
inactivate cytokinins, but the precise in vivo function of these
N-glucosyl-conjugates is unknown (Mok and Mok,
2001).
O-Glucosyl-conjugates of the
N6-side chain are a common modification in
all plants. In addition, O-xylosyl-conjugates have been
reported in Phaseolus spp. (Mok and Mok, 2001). The
purified corresponding glycosyltransferases of Phaseolus
spp. exhibited a high substrate specificity both to the cytokinins
zeatin and dihydrozeatin and to the glycosyl donor (Dixon et al.,
1989). The zeatin-O-glucosyltransferase (ZOG1) strongly
preferred UDP-Glc while the zeatin-O-xylosyltransferase
(ZOX1) exclusively utilized UDP-Xyl. The two genes,
ZOG1 and ZOX1, have been isolated from Phaseolus spp. and the binding site of the UDP-Glc has been
mapped by domain swap experiments (Martin et al., 1999a, 1999b; Mok and Mok, 2001). Recently the isolation of a
cis-zeatin-O-glucosyltransferase (cis-ZOG1) has been
reported that specifically glucosylates cis-zeatin but not trans-zeatin
or dihydrozeatin (Martin et al., 2001). The authors suggested that the
high specificity of cis-ZOG1 to the cis-isomer may indicate an
important role for cis-zeatin in plants.
O-Glycosylated cytokinins are resistant to the cleavage of
the N6-side chain by cytokinin oxidases
(see below). Additionally, these forms can easily be converted into
active cytokinins by 
-glucosidases (Brzobohaty et al., 1993). Thus,
it is believed that O-glycosylated cytokinins are inactive,
stable storage forms that play an important role in balancing cytokinin levels.
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DEGRADATION |
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TOP
INTRODUCTION
BIOSYNTHESIS AND METABOLISM
BIOSYNTHESIS
METABOLISM
DEGRADATION
TRANSPORT
PERCEPTION AND SIGNAL...
CONCLUSION
LITERATURE CITED
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Free cytokinin bases and nucleosides with an unsaturated
N6-side chain are irreversibly degraded by
cleavage of the side chain by cytokinin oxidases. Cytokinin oxidases
are found in many plant species, and they represent a class of highly
diverse proteins (Jones and Schreiber, 1997). The genes of two highly
similar cytokinin oxidases have been isolated from maize (Zea
mays; Houba-Hérin et al., 1999; Morris et al., 1999).
Heterologous expression in Pichia pastoris and
Physcomitrella patens confirmed the predicted cytokinin oxidase activity. Maize cytokinin oxidase and its Arabidopsis homologs contain a signal sequence for the secretory pathway, and they
may be therefore extracellular proteins, but no in vivo localization
studies have yet been reported.
Werner et al. (2001) have recently constructed transgenic tobacco
(Nicotiana tabacum) expressing four Arabidopsis
homologs of the maize cytokinin oxidase from the CaMV 35S promoter. For the first time, this allowed the phenotypic consequences of reduced endogenous cytokinin levels to be determined. Transgenic lines exhibited a higher cytokinin oxidase activity and significantly reduced
amounts of iP and zeatin metabolites, including their glycosides as
compared with wild type. The transgenic lines showed a dwarfed growth
as a consequence of a severely retarded shoot development. In contrast,
the growth of the root system was enhanced, indicating that cytokinin
may have opposing roles in shoot and root development. These defects
could be traced back to alterations in the cell number and the rate of
cell division in the apical meristems. Shoot apical meristems consisted
of fewer cells with sizes comparable to wild type. Leaves were formed
from a significantly decreased number of cells, which was partly
compensated for by an increased cell size. Nevertheless, the transgenic
leaves were about 15% of the size of their wild-type counterparts. The
reduced endogenous cytokinin levels resulted in alterations in cell
proliferation in the apical meristems, consistent with an in vivo role
for cytokinins in the regulation of cell division. In contrast, there
was an increased number and size of cells in the root apical meristem, which contributed to a general enlargement of the root.
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TRANSPORT |
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TOP
INTRODUCTION
BIOSYNTHESIS AND METABOLISM
BIOSYNTHESIS
METABOLISM
DEGRADATION
TRANSPORT
PERCEPTION AND SIGNAL...
CONCLUSION
LITERATURE CITED
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Based on the occurrence of cytokinins in the xylem sap and the
identification of the root tip as a major site of cytokinin biosynthesis, it is generally assumed that cytokinins are transported in the xylem to exert their effects on the aerial parts of plants. A
purine transporter, AtPUP1, has been isolated in Arabidopsis by the
functional complementation of a yeast mutant deficient in adenine
uptake (Gillissen et al., 2000). Adenine uptake was competitively
inhibited by free cytokinin bases. Although no direct evidence for
cytokinin transport was presented, this competitive inhibition suggests
that AtPUP1 represents a possible cytokinin/purine transporter involved
in the translocation of root-derived cytokinins to the aerial plant parts.
Insight into the biological role of cytokinin transport was provided by
feeding roots of nitrogen-depleted maize with nitrate (Takei et al.,
2001b). In response to the applied nitrate, cytokinin accumulated first
in the roots, subsequently in the xylem sap, and finally in leaves.
This suggests that in response to nitrate, these plants made cytokinins
in the root, which was subsequently transported into the aerial portion
of the plant through the xylem. Thus, cytokinin may represent a
long-distance signal for nitrogen availability from the root to the
shoot, presumably to coordinate shoot and root development.
Somewhat conflicting results with respect to the biological
significance of transported cytokinin were obtained from reciprocal grafting experiments with wild-type and cytokinin-overproducing tobacco
plants (Faiss et al., 1997). The phenotypic effects of elevated
cytokinin were restricted to the part of the plant that was derived
from the ipt overexpressing mutant. Elevated level of
cytokinins in the root led to only a slight increase in cytokinin levels in the xylem and had no phenotypic consequences in the scion.
Thus, it was concluded that cytokinin may act as paracrine signal, at
least with respect to apical dominance and leaf senescence.
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PERCEPTION AND SIGNAL TRANSDUCTION |
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TOP
INTRODUCTION
BIOSYNTHESIS AND METABOLISM
BIOSYNTHESIS
METABOLISM
DEGRADATION
TRANSPORT
PERCEPTION AND SIGNAL...
CONCLUSION
LITERATURE CITED
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The concept of cytokinin as a phytohormone implies that the site
of synthesis and the site of action can spatially be separated. Thus,
the search for receptors for cytokinin is almost as old as its
discovery. Biochemical approaches led to the isolation of cytokinin
binding proteins, but none has been unequivocally shown to be a
receptor. The report of a G-protein-coupled receptor, GCR1, as a
candidate for a cytokinin receptor has recently been retracted (Kanyuka
et al., 2001). In an activation-tagging mutagenesis, a His kinase
(CKI1) was identified by its ability to confer cytokinin-independent callus growth when overexpressed in Arabidopsis (Kakimoto, 1996). It
was proposed that the elevated basal level of CKI1 overcomes the
cytokinin requirement for callus growth and greening, and that CKI1
represents a putative cytokinin receptor. Transient expression of CKI1
in Arabidopsis protoplasts indeed showed activation of the promoter of
cytokinin primary response gene (Hwang and Sheen, 2001). However, this
expression of ARR6 was constitutive, not responsive to
exogenous cytokinin. The constitutive induction of ARR6 by
CKI1 in this system might reflect cross talk between CKI1 and the
cytokinin signaling pathway due to the overexpression of CKI1, and may
not reflect the situation in vivo. Although the phenotype of the
CKI1 overexpressing mutant implicates an important role for
CKI1 or a similar His kinase in the regulation of cell proliferation
and/or division, the nature of the mutation (i.e. gain-of-function)
does not allow a definitive conclusion regarding the wild-type function
of the protein.
The involvement of a His kinase in cytokinin signaling has recently
been confirmed by two groups who have reported the identification of a
cytokinin receptor in Arabidopsis. This gene, CRE1, encodes a His kinase that shows weak similarity to the amino acid sequence of
CKI1 (Inoue et al., 2001; Suzuki et al., 2001a). A screen of 19,000 ethyl methanesulfonate-mutagenized Arabidopsis plants
identified a single mutant line that displayed reduced sensitivity to
cytokinin in tissue culture, including shoot formation and
proliferation of green callus (Inoue et al., 2001). Additionally,
cre1 mutant seedlings displayed resistance to cytokinin in a
root elongation assay. An independently isolated T-DNA-tagged allele of
cre1 showed a similar phenotype. Both mutations appear to be
partial loss-of-function alleles by molecular analysis (see also
below), but are semidominant in the root elongation assay. This
semidominance of a hypomorphic allele implies haploinsufficiency,
indicating a dose-dependent action of the receptor in this response.
CRE1 encodes an unusual type of hybrid kinase with two
C-terminal response regulator domains. In addition, CRE1 contains a His
kinase domain and two transmembrane regions flanking a novel presumed
extracellular domain. All identified mutations affect the N-terminal
part of the receptor, and the functional relevance of the two response
regulator domains is unknown.
The phenotype of cre1 mutants and the homology to His
receptor kinases are consistent with a role in cytokinin signaling, but
compelling evidence that cytokinin can indeed act as a ligand for CRE1
was provided by a series of elegant experiments using yeast and
E. coli (Inoue et al., 2001; Suzuki et al., 2001a). The
experiments were based on the idea that endogenous responses can be
influenced by expression of CRE1 in a suitable genetic background of a
heterologous system, and that these responses can be altered in a
cytokinin-dependent manner. In yeast, the sensor His kinase SLN1, which
is involved in osmosensing, inhibits the activity of SSK2 (a
mitogen-activated protein kinase kinase kinase) by a
phosphorelay consisting of SLN1 itself, the His transfer protein YPD1,
and the response regulator SSK1 (Fig. 2A;
Posas et al., 1998). In wild-type yeast, unphosphorylated SSK1 enables activation of SSK2 by autophosphorylation. Under normal osmotic conditions, this activation is inhibited by phosphorylation of SSK1 by
SLN1 and YPD. Deletion of SLN1 is lethal due to the overactivation of
SSK2 by unphosphorylated SSK1. Viability can be restored by the
expression of the phosphatase PTP2 that inhibits SSK2. Thus, a yeast
strain deleted in SLN1 and carrying the PTP2- gene under the control of
a Gal-inducible promoter is conditionally lethal, allowing growth only
in the presence of Gal. In the absence of Gal, the expression of CRE1
suppressed the conditional lethality, but only when cytokinins were
present in the medium. The cytokinin-dependent rescue by CRE1 required
YPD1, indicating that CRE1 acts through the endogenous yeast
phosphotransfer mechanism. Furthermore, only cytokinins that exhibit
high activity in bioassays, such as trans-zeatin, or benzyladenine,
could suppress lethality on Glc-containing media; the weakly active
cis-zeatin was ineffective (Inoue et al., 2001). Interestingly,
thidiazuron, a N,N'-diphenylurea (DPU)-type
cytokinin, also activates CRE1 in this assay. DPU-type cytokinins
strongly inhibit cytokinin oxidase activity in vitro, and this has been postulated as their mode of action in vivo (Jones and Schreiber, 1997).
However, the activity of thidiazuron in the yeast assay indicates that
DPU-type cytokinins likely also exert their in vivo effects by direct
stimulation of the receptor. This is quite remarkable given the
profound structural differences between DPU- and zeatin-type
cytokinins. Recent results from heterologous expression of CRE1 in
yeast have confirmed that cytokinin does bind directly to CRE1 with
high affinity (Yamada et al., 2001).
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What is the biological role of CRE1? Surprisingly, cre1 has
been shown to be allelic to the wooden leg (wol)
mutation, which causes defects in the development of vascular tissue
(Mähönen et al., 2000). wol seedlings have a
reduced number of cells in the vascular cylinder of the root and the
lower part of the hypocotyl, and these consist almost entirely of xylem
cells (Scheres et al., 1995). Thus, phloem and metaxylem are absent in
these parts of wol. The defects in wol can be
traced back to the late torpedo stage of the embryo, which has a
reduced number of vascular initials due to the lack of specific
asymmetric cell divisions (Scheres et al., 1995; Mähönen et
al., 2000). The absence of phloem and metaxylem cells is presumably
caused by reduced cell division rather than aberrant cell specification
because the cre1 mutant phenotype can be suppressed by the
fass mutation, which has supernumerary cell layers (Scheres
et al., 1995). It was proposed that the determination of xylem cells
precedes the specification of phloem cells and that the reduced number
of initials are completely used up as xylem precursors in
wol, leaving no initials left to specify phloem (Scheres et
al., 1995). This model of the wol phenotype nicely brings
together the role of CRE1/WOL as a cytokinin receptor and the
stimulatory effects of cytokinin on cell division. However, CRE1/WOL
may also have additional roles in the differentiation of vascular
cells. wol has a substantially stronger phenotype than the
other cre1 alleles (Mähönen et al., 2000; Inoue
et al., 2001), although it is unknown if it is a null allele.
Interestingly, the wol mutation is a missense mutation
affecting the extracellular domain of the protein, which is the
putative ligand-binding site (Mähönen et al., 2000). In
vitro binding assays have recently shown that the wol
mutation disrupts binding of cytokinin to CRE1 (Yamada et al.,
2001).
CRE1/WOL is expressed primarily in the root, and
in the embryo it is restricted to the provascular tissue from the root
to the shoulder region of the cotyledons (Mähönen et al.,
2000). This expression pattern may explain the rather unexpected
absence of a phenotype in the aerial portions of wol mutant
seedlings and adults. In contrast to the lack of a shoot phenotype in
intact plants, cre1 mutants show a strong reduction in the
regeneration of shoots from tissue culture cells in response to
cytokinin. It may be that the formation of a shoot in tissue culture
requires the elaboration of a proper vasculature. In this context, it
is interesting that regenerated roots from wol callus
exhibit identical phenotypic aberrations like roots of wol
seedlings (Scheres et al., 1995). Alternatively, CRE1 may be
redundant with other genes in the shoots of intact plants, but not with
respect to shoot formation in vitro. Indeed, there are two closely
related His kinases (AHK2 and AHK3) in Arabidopsis that are mainly
expressed in the aerial parts of wild-type plants (Ueguchi et al.,
2001). Both His kinases are highly homologous to the entire CRE1
sequence, including the putative ligand binding domain, and also share
with CRE1 the presence of two response regulator domains in their C terminus. Furthermore, in a mesophyll protoplast transient expression system, both His kinases may regulate the promoter of a primary cytokinin response gene in a cytokinin-dependent manner (Hwang and
Sheen, 2001; see also below). These results strongly suggest that AHK2
and AHK3 indeed act as cytokinin receptors in Arabidopsis.
The involvement of a hybrid His kinase in cytokinin signaling suggests
that additional homologs of bacterial phosphorelay elements may be
involved in the cytokinin signal transduction pathway. Genes encoding
homologs of these other components, phospho-His transfer proteins and
response regulators, have been identified in Arabidopsis. The possible
role of several of these genes in cytokinin signaling has been
addressed by numerous groups. Recently, for example, the induction of
the promoter of a cytokinin primary response gene, the type-A
ARR6 (see below), was monitored by a reporter in Arabidopsis
mesophyll protoplasts that transiently expressed wild-type and mutated
forms of these genes (Hwang and Sheen, 2001). The results, along with
numerous findings of other groups, provide important and valuable
insights into the putative hardwiring of cytokinin signaling
components. The data for each of the three types of components acting
downstream of the receptors will first be discussed separately, then we
will integrate the information about these components into a working
hypothesis of cytokinin signal transduction.
There are five Arabidopsis genes encoding putative His phosphotransfer
proteins (AHPs), which function as a bridge in the primary
phosphotransfer between the Asp residues present in the receiver domain
of the hybrid sensor kinase and the receiver domain of the response
regulator (Suzuki et al., 2000). Suzuki et al. (2001a) established a
heterologous system in E. coli analogous to the
above-described yeast system to verify that CRE1 could act as a
cytokinin receptor, and to implicate the AHPs as downstream targets of
CRE1. The His phosphotransfer protein in E. coli used in
this analysis was YojN, and the cognate response regulator was RcsB.
The activation of RcsB by CRE1 could be quantified by a RcsB-responsive
promoter fused to the reporter LacZ. Coexpression of four different
AHPs differentially inhibited the reporter expression. It was proposed
that the coexpressed AHPs competed with the endogenous YojN for
phosphotransfer by CRE1, consistent with an interaction between that
CRE1 and these AHPs. The different effectiveness of specific AHPs might
reflect specificity of the proposed CRE1-AHP interactions. However, the
individual expression levels of the four AHPs in this system were not
determined, and thus quantitative measurements should be interpreted
carefully. Nuclear localization studies also support the hypothesis
that at least some of the AHPs are involved in cytokinin signaling
(Hwang and Sheen, 2001). It was shown that AHP1 and AHP2, but not AHP5,
transiently translocate from the cytoplasm to the nucleus of
Arabidopsis protoplasts in response to exogenously applied
trans-zeatin. Finally, several groups have reported yeast two-hybrid
interactions between His kinases and AHPs. Taken together, these
reports indicate that at least a subset of AHPs may be involved in
cytokinin signaling. These AHPs likely transmit the signal from its
site of perception, probably the plasma membrane, to the effectors in
the nucleus. Numerous results indicate that these effectors consist, at
least in part, of the third class of components of phosphorelays, the response regulators.
The response regulators in Arabidopsis (ARRs) form a gene family
that includes the type-A and the type-B ARRs (D'Agostino and Kieber,
1999; Imamura et al., 1999). The type-A ARRs are composed solely of a
receiver domain; the type-B ARRs have an output domain fused to the
receiver. This C-terminal output domain has been demonstrated to
activate transcription in yeast and in planta and to bind to DNA in a
sequence-specific manner. The type-B ARRs have been shown to be
nuclear-localized proteins (Lohrmann et al., 1999; Sakai et al., 2000;
Hwang and Sheen, 2001). The steady-state levels of mRNA of most of the
type-A, but not the type-B ARRs are rapidly and specifically elevated
in response to cytokinin (Brandstatter and Kieber, 1998; Taniguchi et
al., 1998; Kiba et al., 1999; D'Agostino et al., 2000). Homologous
response regulators in maize exhibit very similar induction
characteristics indicating that the function of type-A ARRs is highly
conserved in plants (Sakakibara et al., 1998). The induction by
cytokinin of several of the type-A ARRs is due, at least in part, to
the transcriptional activation of these genes and is not blocked by
cycloheximide, which indicates that these are primary response genes
(Brandstatter and Kieber, 1998; D'Agostino et al., 2000). The
similarity to bacterial signaling elements predicted to act downstream
of sensor His kinases, combined with the rapid induction by cytokinin,
suggest that these type-A ARRs may play a role in cytokinin signaling. Consistent with this, the ARR5 gene has been found to be
expressed primarily in the shoot and root apical meristems, likely
sites of cytokinin action (D'Agostino et al., 2000). Recently, studies in protoplasts have shown that the overexpression of type-A ARRs represses their own expression, suggesting that type-A ARRs may provide
a negative feedback regulation of cytokinin signaling (Hwang and Sheen,
2001). Interestingly, mutant forms of these genes lacking a conserved
Asp, the predicted phosphoaccepting site, were equally effective in
their feedback inhibition. Thus, wild-type and mutant proteins may
interact with cytokinin signaling elements, such as the AHPs, making
these components inaccessible to the pathway. Type-A ARRs may also
interact with additional partners and could have dual functions. Their
activation by cytokinin may negatively feedback into cytokinin
signaling, but would also trigger yet unknown, secondary responses to
cytokinin. This model may also explain why there are 10 type-A ARRs in Arabidopsis.
In two recent publications, the transcriptional control of the type-A
ARRs was shown to be mediated, at least partly, by type-B ARRs (Sakai et al., 2001; Hwang and Sheen, 2001). An arr1 (a
type-B ARR) knockout mutant was found to be partially resistant to
cytokinin in shoot regeneration and root elongation assays, and an ARR1 overexpressing transgenic line displayed increased sensitivity to
cytokinin in these assays. These results suggest that this type-B gene
plays a role in cytokinin responsiveness. Examination of the expression
of the type-A genes in the arr1 mutants revealed that the
loss-of-function allele exhibited reduced expression of several type-A
ARR transcripts and that overexpression of ARR1 resulted in
an enhanced induction of the type-A genes, even in the presence of
cycloheximide. These results, coupled with the presence of multiple
ARR1 binding sites in the promoter of several type-A ARRs suggest that
ARR1 directly regulates the transcription of these primary targets of
cytokinin signaling (Sakai et al., 2001). The activity of the ARR1
output domain is inhibited by the N-terminal response regulator domain
(Sakai et al., 2000). Thus, it was proposed that the inhibition of the
transcriptional activator domain by the response regulator domain was
modulated by cytokinin. Similar results were independently obtained by
transient expression studies in Arabidopsis protoplasts (Hwang and
Sheen, 2001). Overexpression of three type-B ARRs, ARR1, ARR2, and
ARR10, highly increased both the basal level and the induction of the promoter of a type-A ARR, ARR6. ARR2 exhibited
the strongest induction and was consequently analyzed in greater
detail. Expression of the DNA binding domain of ARR2 without the
receiver and transactivator domain abolished the cytokinin
responsiveness of the ARR6 promoter, presumably acting as a
dominant negative form. The overexpression of ARR2 in planta promoted
rapid cell proliferation and shoot and leaf formation in tissue
cultures even in the absence of exogenous cytokinin. The transgenic
plants also showed delayed leaf senescence as expected for cytokinin overaction.
These results implicate at least several of the type-B ARRs as positive
regulators of cytokinin responses. Surprisingly, mutated proteins in
the transient assay in which the Asp, the probable site of
phosphorylation, was altered to Asn, were equally effective in the
induction of the ARR6::reporter by cytokinin
(Hwang and Sheen, 2001). This raises the question of whether cytokinin
signaling acts as a classical His-to-Asp phosphorelay, at least with
regard to the induction of the type-A ARR genes. The authors
propose that an endogenous repressor may be liberated by the
overexpression of the type-B ARRs. Several indirect arguments support
the phosphorelay hypothesis. The conservation of the presumed
phosphorylation sites within all 10 type-A and about 12 type-B ARRs in
Arabidopsis as well as the conservation of these sites between maize,
rice (Oryza sativa), and Arabidopsis homologs
strongly suggests the functional relevance of these sites. At least at
the level of the receptor, the phosphorelay hypothesis is also
experimentally verified. The cytokinin-dependent complementation in
yeast and E. coli requires an interaction of CRE1 with
endogenous His transfer proteins similar to their interaction with
their cognate His kinases. In addition, mutant forms of CRE1 in which
these sites have been altered are unable to induce the
ARR6::reporter in protoplasts by application of
cytokinin. Thus, it is highly likely that CRE1 acts both in the
transient expression system and in the cytokinin-dependent complementation in yeast and E. coli as a His kinase.
Biochemical studies and loss-of-function mutants of the AHPs and ARRs
should enable us to clarify the significance of their presumed
phosphorylation sites.
These results are consistent with the following model of cytokinin action. Binding of cytokinin to CRE1 (and possibly other related His kinases) initiates a phosphorelay that culminates, via an AHP intermediate, in the phosphorylation and activation of a set of the type-B ARR(s). These then activate the transcription of the type-A genes, which in turn negatively feedback into the pathway. The type-A ARRs, perhaps together and/or in parallel with the activated type-B ARRs, interact with various effectors to mediate the changes in cell function appropriate to cytokinin (Fig. 2B). The near future should see significant refinement of this model as the tools are now in hand to analyze the interactions among these elements.
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CONCLUSION |
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TOP
INTRODUCTION
BIOSYNTHESIS AND METABOLISM
BIOSYNTHESIS
METABOLISM
DEGRADATION
TRANSPORT
PERCEPTION AND SIGNAL...
CONCLUSION
LITERATURE CITED
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The field of cytokinin research has seen dramatic advancements during the last 2 years. Several of the key elements of cytokinin action and metabolism have been identified, but many questions need to be addressed. The in vivo role of most of the members of the phosphorelay in Arabidopsis remains unknown, including their putative function in cytokinin signaling. The function and specificity of the interactions among the various components in vivo represents one of the key questions. The analysis of expression patterns and the identification of mutants in these genes will enable us to understand the numerous effects of cytokinin on plant physiology and development at the molecular level. Furthermore, these genes can now be used to identify additional regulators and downstream targets, which should help unveil the complex network of cytokinin metabolism and signaling.
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FOOTNOTES |
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* Corresponding author; e-mail jkieber{at}unc.edu; fax 919- 962-1625.
1 This work was supported by the National Science Foundation (grant no. MCB-9996354 to J.J.K.).
www.plantphysiol.org/cgi/doi/10.1104/pp.010773.
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LITERATURE CITED |
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TOP
INTRODUCTION
BIOSYNTHESIS AND METABOLISM
BIOSYNTHESIS
METABOLISM
DEGRADATION
TRANSPORT
PERCEPTION AND SIGNAL...
CONCLUSION
LITERATURE CITED
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