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492
The retinoblastoma pathway in plant cell cycle and development
Crisanto Gutierrez
The activity of cyclin-dependent kinases (CDKs) on specific
targets mediates the temporal regulation of plant cell cycle
transitions. The sequential activity of CDKs and the spatial
regulation of cell proliferation during plant development,
however, are still poorly understood. Understanding these
aspects depends on the identification of the downstream
targets and upstream modulators of CDKs and their regulation
in response to mitogenic and/or differentiation signals. Current
efforts to elucidate the answers to these questions are very
promising; in particular, recent works reveal the essential role
that the retinoblastoma pathway plays in controlling cell cycle
progression and, presumably, some developmental events.
The existence of an analogous pathway in plants has been
a matter of speculation until recently since none of the
components had been identified. Advances in the study of
G1/S regulators in plants, however, have indicated that a
Rb-like pathway might operate in regulating plant cell
growth. Surprisingly, to some extent, the components
identified so far are similar to some of those functioning in
mammalian cells and are, therefore, totally unrelated to
those found in yeast. Recent efforts to understand G1/S
regulation in plant cells, with particular emphasis on the
Rb pathway, will be the subject of this review.
Plant Rb protein(s)
Addresses
Centro de Biología Molecular ‘Severo Ochoa’, Consejo Superior de
Investigaciones Científicas (CSIC) – Universidad Autónoma de Madrid
(UAM), Cantoblanco, 28049 Madrid, Spain;
e-mail: cgutierrez@trasto.cbm.uam.es
Current Opinion in Plant Biology 1998, 1:492–497
http://biomednet.com/elecref/1369526600100492
© Current Biology Ltd ISSN 1369-5266
Abbreviations
CAK
CDK-activating kinase
CDK
cyclin-dependent kinase
Rb
retinoblastoma
RbIP
Rb-interacting protein
STF
S-phase-specific transcription factor
UTR
untranslated region
Introduction
When cell responses are integrated at a higher organizational level (organ, organism), the differences between
plants and animals become very apparent (for example
body organization and development). The lack of cell
migration, the existence of a continuous morphogenetic
process and the plasticity of the plant cell lead to a requirement for continuous cell proliferation and the existence of
highly regulated gene expression programs. In spite of
these and other differences, the overall strategy which
evolved in plant cells to regulate the events required to
complete the cell division cycle seems to be similar to that
in other eukaryotes, as these events are mediated by the
action of cyclin-dependent kinases (CDKs) associated with
cyclins [1,2,3•]. In mammalian cells, members of the
retinoblastoma (Rb) family, also known as ‘pocket’ proteins,
are crucial to control the passage of cells through G1 and
G1/S, and the reactivation of G0 cells [4]. Rb operates as a
tumor suppressor and as a negative regulator of cell proliferation. Three members comprise the Rb family, namely
the Rb tumor suppressor, and the p107 and p130 proteins.
Their function depends on the association with transcription factors of the E2F family [5]. These complexes are
disrupted by sequential phosphorylation of the Rb moiety
carried out by various cyclin-CDK complexes [6,7].
The hypothesis that a Rb-mediated pathway may exist in
plants received initial support from two lines of evidence:
first, the isolation of homologues of D-type cyclins from
Arabidopsis [8] and alfalfa [9], which contain an LXCXE
amino acid motif known to mediate Rb-binding in human
cells; and second, the identification of proteins in geminiviruses (a group of plant DNA viruses with small
genomes) that also contained the LXCXE motif required
for efficient viral replication in cultured cells [10]. This,
together with their absolute requirement for cell factors to
complete the viral replication cycle, makes geminiviruses
useful tools in plant cell cycle and DNA replication studies.
Early in the development of this field, a maize EST [11] was
used to isolate cDNA clones encoding proteins with homology to human Rb family proteins from cDNA libraries made
from maize endosperm (ZmRb, [12]) and two week old
seedlings (ZmRb1, [13]). Several ZmRb1 cDNA clones, differing in their 3′-untranslated region (3’UTR), were isolated
(Q Xie and C Gutierrez, unpublished data). Analysis by
Northern blot and rapid amplication of 5′ cDNA ends polymerase chain reaction in maize cells indicated the existence
of mRNAs of a size consistent with that of the longest
ZmRb1 cDNA isolated [13]. In due course, two classes of
cDNAs encoding Rb-related proteins from two maize
genes, RRB1 and RRB2, were isolated [14•]. The two
longest RRB1 cDNAs also differ in their 3′UTR and have
the potential to encode a nuclear protein of 866 amino acids,
whereas the RRB2 cDNAs seem to represent partial clones
[14•]. Interestingly, RRB transcripts are abundant in the
shoot apex, but barely detected in 2-week seedlings. Thus,
it is possible that expression of different members of the Rb
protein family might be spatially and/or temporally regulated. Data on the genomic structure of the maize Rb gene(s),
on Rb from other plant species, and on the functional role of
Rb should contribute significantly to the field.
Structural conservation between plant and
animal Rb proteins
Amino acid sequence comparison has revealed a striking
conservation of the domain organization of Rb family
The Rb pathway in plant cell cycle and development Gutierrez
493
members, including plants. They contain the domains A
and B (50–65% similarity), which together form the socalled A/B pocket, flanked by carboxy- and amino-terminal
domains. All family members, apart from Drosophila, have
a spacer of variable length between the A and B domains
(Figure 1). Whether plant Rb is functionally equivalent to
any of the human Rb family members or whether it shares
some or all of their cell cycle regulatory properties remain
to be determined. The conservation among pocket proteins does not occur merely at the level of their primary
sequence. The crystal structure of the human A/B pocket
domain [15] has revealed that the three-dimensional structure might also be conserved, in particular, for two clusters
of surface residues. One is the LXCXE-binding site, located within the B domain and consisting of a hydrophobic
groove bordered by a positively charged rim (Figure 2).
This explains the similarity in Rb-binding properties
between animal virus oncoproteins, such as Simian
Virus 40 large T antigen, and plant geminivirus Rb-binding proteins [10,14•]. The other is located in the interface
between the A and B domains. Interestingly, it is in these
two clusters that mis-sense mutations frequently map in
human tumors [16]. Thus, an exciting research topic for
the future will be to identify the phenotypic consequences
that mutations in these conserved clusters in Rb could
have on plant cell cycle, growth and development.
Furthermore, the D-type cyclins are crucial players in cell
cycle regulation as high levels of cycD1 are frequently
found in human tumors and cycD1 overexpression promotes hyperplasia and adenocarcinomas [17]. Although
similar experiments are not available yet in plants, ectopic
expression of the mitotic cyclin cyc1At stimulates growth
without altering root development, and does not result in
neoplasia [18].
that regulation of Rb activity depends on its phosphorylation level [13]. The cluster in the carboxy-terminal
domain, known to be phosphorylated by different
CDK/cyc complexes in human cells [19], could be of particular relevance. Thus, it is conceivable, by analogy to
animal cells, Rb phosphorylation by one or more CDK/cyc
complexes allows the release of Rb-bound S-phase-specific transcription factors (STFs).
Rb as a CDK substrate
Future studies should focus on the identification of pathways regulating the activity of CDK/cyc complexes.
Steps towards this goal have been taken by the isolation
of the Arabidopsis cak1At gene encoding a CDK-activating
Plants contain an abundant CDK gene family [3•]. The
presence of consensus CDK phosphorylation sites
(defined by the amino acid motif S/TP), strongly suggests
The search for the CDK/cyc complex(es) acting on plant
Rb is intense in a number of laboratories although, so far,
their identification has been elusive. One could anticipate that such activity should be present during the G1
and S phases. Some CDK activities have been characterized in Arabidopsis [20] and in alfalfa cells [21••],
although it remains to be determined whether they can
functionally use Rb as a substrate. Complementary in
vitro phosphorylation studies using maize Rb and human
CDK/cyc complexes have shown that plant Rb can be
readily phosphorylated by human CDK4/cycD,
CDK2/cycA and CDK2/cycE complexes [22 ••].
Moreover, an intact pocket domain is required for efficient phosphorylation by CDK4/cycD, as in human cells,
but not by the other complexes [22••]. Although the
functional significance of these studies still needs to be
addressed, it is conceivable that one (or more) plant
CDK complexed to cycD and/or cycA, functionally
equivalent to human CDK4/cycD and CDK2/cycA, regulates plant Rb function by sequential phosphorylation.
In this context, Rb has been also implicated in S phase
regulation in the absence of an intervening mitosis, as an
S-phase-associated kinase activity is increased in maize
endosperm, coinciding with the initiation of endoreduplication [12,23].
Figure 1
Comparison of plant and animal Rb family
members. The tree is based on amino acid
homologies of the region of each protein
containing the A/B pocket domain (black
boxes) and the spacer domain, using the
PILEUP program of the GCG package. Bars
at the right represent members of each
subgroup to show the overall conservation of
their domain organization. Note that the
sequences of RRB1 and ZmRb1 differ in
some residues, that the maize RRB2b has a
different carboxy-terminus and that Drosophila
RBF lacks the spacer domain. The accession
numbers of the sequences used in this study
are: AF007793, X98923, AF007795,
M26391, P33568, M15400, U00113,
A44879, Y09226, L14812, Q64701,
S67171, U50850 and X96975.
Maize RRB1
Maize ZmRb1
Maize RRB2b
Mouse
Rat
Human
Rb
Chicken
Xenopus
Newt
Human
p107
Mouse
Human
p130
Mouse
Drosophila RBF
A
B
Current Opinion in Plant Biology
494
Cell biology
Figure 2
Maize Rb
Human Rb
NEKCADVTIHIFFSKILKLAAIRIRNLCERVQCVEQ.TERVYNVFKQILEQQTT.LFFNRHIDQLILCCL
PLKSTSLSLFYKKVYRLAYLRLNTLCERLLSEHPELEHIIWTLFQHTLQNEYELMRDRHLDQIMMCSM
α11
α12
cyc fold α1
α13
α14
cyc fold α3
cyc fold α2
x
Tumor-derived mutations in human cells
x
YGVAKVCQLELTFREILNNYKREAQCKPEVFSSIYIGSTNRNGVLVSRHVGIITFYNEVFVPAAKPFLVSL
YGICKVKNIDLKFKIIVTAYKDLPHAVQETFKRVLI........KEEEYDSIIVFYNSVFMQRLKTNILQY
α15
cyc fold α4
The groove
LxCxE
binding
site
The rim
+
+
α16
+
+
β1
β2
α17
+
α18
cyc fold α5
+
Current Opinion in Plant Biology
Amino acid conservation and predicted structure of domain B of maize
Rb derived from the crystal structure of the A/B pocket of human Rb
[15]. Domain B contains eight α-helices (α11 through α18), five of
which participate in the so-called ‘cyclin fold’ (α-helices cycfold α1
through cycfold α5). The two short β strands (β1 and β2) are also
shown. Note that the eight amino acid insertion present in maize Rb is
predicted to be located in a loop between the β strands. Conserved
residues are shown in boldface: gray background, the LXCXE binding
groove (hydrophobic amino acids); black background, the rim
(positively charged amino acids), and empty boxes, the residues
frequently mutated in human tumors (X).
kinase (CAK) which is able to complement CAK mutations in yeasts and to specifically activate human CDK2
[24••]. Another plant gene with significant homology to
CAKs is the rice R2 gene whose expression is up-regulated in meristems and part of the elongation zone in
gibberellin-treated rice internodes, consistent with its
potential role in G1/S progression [25•]. On the other
side, the identification of other CDK-interacting proteins
such as a homolog of the yeast p13suc1 gene from
Arabidopsis [26] and an Arabidopsis CDK inhibitor (ICK1),
related to human p27 protein, which interacts with both
cdc2a and cycD3 and is induced by abscisic acid [27•]
should contribute significantly to our understanding of
the CDK regulatory network.
changes of the motif, for example LXCXK, drastically
reduce or abolish Rb-binding [13], whereas others, for
example LXCXQ, do not have such strong effects (L Liu
and J Stanley, personal communication). In Mastreviruses,
the Rep protein, required for viral DNA replication, is
translated from the same RepA-encoding mRNA after a
splicing event [29]. This Rep protein contains an LXCXE
motif but, interestingly, it does not bind to plant Rb ([28];
L Liu and J Stanley, personal communication; Q Xie and
C Gutierrez, unpublished data). Presumably, the LXCXE
motif is hidden since a carboxy-terminal deleted version of
WDV Rep can bind to plant Rb (Q Xie and C Gutierrez,
unpublished data). Second, the three Arabidopsis D-type
cyclins bind to maize Rb [22••]. Interaction between RRB1
and cycD3 in yeast seems to be enhanced in a carboxy-terminal deleted cycD3 protein [14•].
A broad spectrum of plant Rb-interacting
proteins
Since the isolation of cDNAs encoding plant Rb proteins, the identification of Rb-interacting proteins
(RbIPs) has allowed us to take a glimpse at the potential
roles of different Rb-containing complexes in plants.
RbIPs belong to two groups according to the domain
mediating binding to Rb.
LXCXE-containing RbIPs
First, as already mentioned, subgroup I geminiviruses
(Mastreviruses) encode a RepA protein that binds plant Rb
protein through its LXCXE motif. Examples are the wheat
dwarf virus (WDF; [13]), the maize streak virus (MSV;
[28]) or the bean yellow dwarf virus (BYDV; L Liu and
J Stanley, personal communication) RepA protein. Some
LXCXE-lacking RbIPs
Geminiviruses from the other two subgroups (II or
Curtoviruses and III or Begomoviruses), do not encode any
protein homologous to Mastrevirus RepA but they all encode
a Rep protein. Interestingly, Rep protein of tomato golden
mosaic virus, a Begomovirus, lacks an LXCXE motif but still
binds to maize RRB1 [14•]. Whether this represents a difference in the strategy used by different geminiviruses to
impinge on the Rb pathway is an open question. A second
set of proteins, related to the S. cerevisiae MSI1 protein [30]
and the human RbAp46 and RbAp48 proteins [31], has been
recently isolated from tomato (LeMSI1) and from
Arabidopsis (AtMSI1-3) [32•]. Their function is unknown,
but the human counterparts have been implicated in
The Rb pathway in plant cell cycle and development Gutierrez
processes related to chromatin remodelling [33,34].
Assuming similar roles in plants, it is conceivable that they
have implications in plant growth, morphogenesis and development. Third, a member of the E2F transcription factor
family has recently been cloned in plants [35•] and, like the
animal E2Fs, also lacks a LXCXE motif.
In addition, an increasing number of other proteins are
known to interact with Rb in human cells [36]. It is
expected, therefore, that more plant RBIPs will be identified in the near future. Among them, some could belong
to the family of human histone deacetylases which contribute to Rb-mediated transcriptional repression [37,38].
Others might be related to human oncogenes, differentiation and development factors. Still, it may occur that novel
RBIPs, unique to plants, may have evolved as a consequence of the specific demands of the plant body
organization and physiology.
the basal meristematic part and a progressive increase in differentiated cells along the leaf. In this differentiated zone,
Rb-immunoreactive ~110 kDa proteins accumulate, whereas
they are barely detected (or absent) in the basal zone [22••].
Plant E2F mRNA can be detected in proliferating as well as
in differentiated cells [35•]. These studies, together with the
increasing evidence of the role of cell proliferation, and perhaps cell cycle regulators, in plant development [46] suggest
a role for the components of the Rb pathway at some stages
during plant morphogenesis and patterning.
Conclusions and prospects
A summary of the current knowledge of the Rb pathway
in plants is shown in Figure 3. The picture is far from
Figure 3
Hormones/Nutrients
Regulation of G1/S-specific gene expression
by Rb-bound transcription factors
The correct passage of cells throughout the cell cycle
requires a strict temporal control of transcriptional activity
([3•] and references therein). For some genes, such as
those encoding ribonucleotide reductase, histones, and
cyclin D3, an up-regulation at G1/S and S phases has been
found [39–41]. The molecular nature of S-phase-specific
transcription factors, however, is not known. In human
cells, expression of G1/S genes frequently depends on
E2F transcription factors whose activity is modulated by
binding to Rb family members [5].
Auxin
Cytokinin
Sucrose
CDK
CycD3
CycD2
Geminiviruses
Rb - binding
proteins
The Rb pathway in plant differentiation and
development
Components of the Rb pathway are involved not only in cell
cycle control but also in mammalian [42,43] and Drosophila
development [44,45]. Rb also seems to play a role in growth
and development in plants. Thus, in maize leaves, a proliferative gradient exists with a high amount of dividing cells in
Negative
signals
CycD - CDK ?
CAK
?
P
CycD - CDK
?
CKI
E2F
DP ?
Rb
Is G1/S specific gene expression in plants mediated by
E2F-like proteins? The following evidence strongly suggests that this is the case: the ribonucleotide reductase and
cyclin D3 promoters, but not those of histone genes, contain binding sites similar to those of human E2F (C Gigot,
personal communication; J Murray, personal communication); the structural conservation of the A/B pocket
proteins [15]; maize Rb interacts, although weakly, with
human E2F-1 in yeast [14•,22••,35•]; and finally, maize Rb
represses E2F-dependent transcription in human cells
[22••]. As the identification of plant E2F-like protein(s)
and/or gene(s) has been so elusive to date, a significant
step ahead has been the recent isolation of a cDNA encoding a plant E2F [35•] which has a domain organization
similar that of the human E2F-1 subset of proteins. The
identification of this novel component of the Rb pathway
will allow further studies on its expression pattern and
transcriptional activity related to Rb binding.
495
Rb
P
E2F
Rb
DP ?
?
Differentiated
state
Current Opinion in Plant Biology
G1 / S
Transition
A proposal for the regulation of the Rb pathway. The key component of
the pathway is the complex constituted by Rb, E2F and DP proteins,
the latter still to be identified. Plant Rb contains several potential CDKphosphorylation sites (S/TP), in particular a carboxy-terminal cluster that
may be phosphorylated by CDK/cyc complexes, for example,
CDK/cycD. Changes in the phosphorylation state of Rb, and
presumably of its partners, could modulate their regulatory functions.
The release of the Rb-bound E2F/DP factors after Rb phosphorylation
by activated CDK/cycD would regulate genes required at the G1/S
transition. It should be kept in mind that up-regulation at G1/S may also
occur through other mechanisms. CDK is activated by specific
phosphorylation by CAKs and its function inhibited by specific inhibitors
(CKI). It is tempting to speculate that the expression of these G1/S
regulators and/or their activities may be regulated by hormones,
nutrients and/or negative growth signals. The normal Rb
phosphorylation pathway may be bypassed in geminivirus-infected cells
by the action of virally encoded Rb-binding proteins. The presence of
Rb and E2F messages in differentiated tissues suggests that, in
addition to their role during the cell cycle, Rb and E2F/DP complexes
may be crucial for the differentiation state of plant cells. These putative
interactions have not been demonstrated yet to occur in vivo.
496
Cell biology
complete, as many questions remain unanswered. On the
basis of recent results, however, the field is moving fast
and in extremely attractive directions. First, we need to
identify, clone and characterize at the molecular level
other components of the pathway. Second, since disruption of G1/S regulators in mammals leads to
transformation and tumor development, similar studies in
plants will serve to explain why they are extremely refractory to neoplastic transformation. Third, the normal G1/S
events seem to be altered by geminivirus infection as upregulation of genes, normally switched off in
differentiated cells, occurs. One possibility is that this
and/or other cellular effects of geminivirus proteins might
be a consequence of the interference with the Rb pathway. If this is the case, an abnormal S-phase, or at least
some S-phase functions and perhaps cellular DNA replication, could be triggered. Fourth, if it is proven that
Rb/E2F complexes participate in regulating differentiation decisions, a complete new window will be open to
look at plant development. These are just a few of many
aspects currently being investigated. Thus, we look forward to new and exciting results in the near future.
Acknowledgements
I thank D Dudits, C Gigot (deceased), H Hirt, D Inze, J Murray and J
Stanley for sharing ideas and communicating unpublished results, E
Martinez-Salas for comments on the manuscript, and past and present
members of the lab for contributing with their work, discussions and
stimulating environment. The work is partially supported by grants PB960919 (DGES), CI1*-CT94-0079 (European Union) and 06G/046/96
(Comunidad de Madrid) and by an institutional grant from Fundación
Ramón Areces.
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is induced by abscisic acid. Plant J 1998, 15:501-510.
A study of ICK1, a CDK inhibitor related to human p27Kip1 protein, in terms
of gene structure, interaction with both A. thaliana Cdc2a and CycD3, and
induction by the plant growth regulator abscisic acid.
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•
28. Horvath GV, Pettko-Szandtner A, Nikovics K, Bilgin M, Boulton M,
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This is the first report of the isolation of a cDNA encoding a plant E2F protein
by yeast two-hybrid screening using ZmRb1 as a bait. It identifies a unique
Rb-binding motif, evaluates its binding properties to different Rb family members and studies plant E2F expression in proliferating and differentiated cells.
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•
proteins binds to the retinoblastoma protein in both plants and
animals. Plant Cell 1997, 9:1595-1606.
This paper reports the cloning by PCR using degenerate oligonucleotides of
plant proteins related to yeast MSI1 and human RbAp46 and RbAp48 proteins. The plant proteins bind to maize RRB1 and to an ~65 kDa protein of
unknown identity.
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plant E2F transcription factor reveals unique and conserved
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The retinoblastoma pathway in plant cell cycle and development
Crisanto Gutierrez
The activity of cyclin-dependent kinases (CDKs) on specific
targets mediates the temporal regulation of plant cell cycle
transitions. The sequential activity of CDKs and the spatial
regulation of cell proliferation during plant development,
however, are still poorly understood. Understanding these
aspects depends on the identification of the downstream
targets and upstream modulators of CDKs and their regulation
in response to mitogenic and/or differentiation signals. Current
efforts to elucidate the answers to these questions are very
promising; in particular, recent works reveal the essential role
that the retinoblastoma pathway plays in controlling cell cycle
progression and, presumably, some developmental events.
The existence of an analogous pathway in plants has been
a matter of speculation until recently since none of the
components had been identified. Advances in the study of
G1/S regulators in plants, however, have indicated that a
Rb-like pathway might operate in regulating plant cell
growth. Surprisingly, to some extent, the components
identified so far are similar to some of those functioning in
mammalian cells and are, therefore, totally unrelated to
those found in yeast. Recent efforts to understand G1/S
regulation in plant cells, with particular emphasis on the
Rb pathway, will be the subject of this review.
Plant Rb protein(s)
Addresses
Centro de Biología Molecular ‘Severo Ochoa’, Consejo Superior de
Investigaciones Científicas (CSIC) – Universidad Autónoma de Madrid
(UAM), Cantoblanco, 28049 Madrid, Spain;
e-mail: cgutierrez@trasto.cbm.uam.es
Current Opinion in Plant Biology 1998, 1:492–497
http://biomednet.com/elecref/1369526600100492
© Current Biology Ltd ISSN 1369-5266
Abbreviations
CAK
CDK-activating kinase
CDK
cyclin-dependent kinase
Rb
retinoblastoma
RbIP
Rb-interacting protein
STF
S-phase-specific transcription factor
UTR
untranslated region
Introduction
When cell responses are integrated at a higher organizational level (organ, organism), the differences between
plants and animals become very apparent (for example
body organization and development). The lack of cell
migration, the existence of a continuous morphogenetic
process and the plasticity of the plant cell lead to a requirement for continuous cell proliferation and the existence of
highly regulated gene expression programs. In spite of
these and other differences, the overall strategy which
evolved in plant cells to regulate the events required to
complete the cell division cycle seems to be similar to that
in other eukaryotes, as these events are mediated by the
action of cyclin-dependent kinases (CDKs) associated with
cyclins [1,2,3•]. In mammalian cells, members of the
retinoblastoma (Rb) family, also known as ‘pocket’ proteins,
are crucial to control the passage of cells through G1 and
G1/S, and the reactivation of G0 cells [4]. Rb operates as a
tumor suppressor and as a negative regulator of cell proliferation. Three members comprise the Rb family, namely
the Rb tumor suppressor, and the p107 and p130 proteins.
Their function depends on the association with transcription factors of the E2F family [5]. These complexes are
disrupted by sequential phosphorylation of the Rb moiety
carried out by various cyclin-CDK complexes [6,7].
The hypothesis that a Rb-mediated pathway may exist in
plants received initial support from two lines of evidence:
first, the isolation of homologues of D-type cyclins from
Arabidopsis [8] and alfalfa [9], which contain an LXCXE
amino acid motif known to mediate Rb-binding in human
cells; and second, the identification of proteins in geminiviruses (a group of plant DNA viruses with small
genomes) that also contained the LXCXE motif required
for efficient viral replication in cultured cells [10]. This,
together with their absolute requirement for cell factors to
complete the viral replication cycle, makes geminiviruses
useful tools in plant cell cycle and DNA replication studies.
Early in the development of this field, a maize EST [11] was
used to isolate cDNA clones encoding proteins with homology to human Rb family proteins from cDNA libraries made
from maize endosperm (ZmRb, [12]) and two week old
seedlings (ZmRb1, [13]). Several ZmRb1 cDNA clones, differing in their 3′-untranslated region (3’UTR), were isolated
(Q Xie and C Gutierrez, unpublished data). Analysis by
Northern blot and rapid amplication of 5′ cDNA ends polymerase chain reaction in maize cells indicated the existence
of mRNAs of a size consistent with that of the longest
ZmRb1 cDNA isolated [13]. In due course, two classes of
cDNAs encoding Rb-related proteins from two maize
genes, RRB1 and RRB2, were isolated [14•]. The two
longest RRB1 cDNAs also differ in their 3′UTR and have
the potential to encode a nuclear protein of 866 amino acids,
whereas the RRB2 cDNAs seem to represent partial clones
[14•]. Interestingly, RRB transcripts are abundant in the
shoot apex, but barely detected in 2-week seedlings. Thus,
it is possible that expression of different members of the Rb
protein family might be spatially and/or temporally regulated. Data on the genomic structure of the maize Rb gene(s),
on Rb from other plant species, and on the functional role of
Rb should contribute significantly to the field.
Structural conservation between plant and
animal Rb proteins
Amino acid sequence comparison has revealed a striking
conservation of the domain organization of Rb family
The Rb pathway in plant cell cycle and development Gutierrez
493
members, including plants. They contain the domains A
and B (50–65% similarity), which together form the socalled A/B pocket, flanked by carboxy- and amino-terminal
domains. All family members, apart from Drosophila, have
a spacer of variable length between the A and B domains
(Figure 1). Whether plant Rb is functionally equivalent to
any of the human Rb family members or whether it shares
some or all of their cell cycle regulatory properties remain
to be determined. The conservation among pocket proteins does not occur merely at the level of their primary
sequence. The crystal structure of the human A/B pocket
domain [15] has revealed that the three-dimensional structure might also be conserved, in particular, for two clusters
of surface residues. One is the LXCXE-binding site, located within the B domain and consisting of a hydrophobic
groove bordered by a positively charged rim (Figure 2).
This explains the similarity in Rb-binding properties
between animal virus oncoproteins, such as Simian
Virus 40 large T antigen, and plant geminivirus Rb-binding proteins [10,14•]. The other is located in the interface
between the A and B domains. Interestingly, it is in these
two clusters that mis-sense mutations frequently map in
human tumors [16]. Thus, an exciting research topic for
the future will be to identify the phenotypic consequences
that mutations in these conserved clusters in Rb could
have on plant cell cycle, growth and development.
Furthermore, the D-type cyclins are crucial players in cell
cycle regulation as high levels of cycD1 are frequently
found in human tumors and cycD1 overexpression promotes hyperplasia and adenocarcinomas [17]. Although
similar experiments are not available yet in plants, ectopic
expression of the mitotic cyclin cyc1At stimulates growth
without altering root development, and does not result in
neoplasia [18].
that regulation of Rb activity depends on its phosphorylation level [13]. The cluster in the carboxy-terminal
domain, known to be phosphorylated by different
CDK/cyc complexes in human cells [19], could be of particular relevance. Thus, it is conceivable, by analogy to
animal cells, Rb phosphorylation by one or more CDK/cyc
complexes allows the release of Rb-bound S-phase-specific transcription factors (STFs).
Rb as a CDK substrate
Future studies should focus on the identification of pathways regulating the activity of CDK/cyc complexes.
Steps towards this goal have been taken by the isolation
of the Arabidopsis cak1At gene encoding a CDK-activating
Plants contain an abundant CDK gene family [3•]. The
presence of consensus CDK phosphorylation sites
(defined by the amino acid motif S/TP), strongly suggests
The search for the CDK/cyc complex(es) acting on plant
Rb is intense in a number of laboratories although, so far,
their identification has been elusive. One could anticipate that such activity should be present during the G1
and S phases. Some CDK activities have been characterized in Arabidopsis [20] and in alfalfa cells [21••],
although it remains to be determined whether they can
functionally use Rb as a substrate. Complementary in
vitro phosphorylation studies using maize Rb and human
CDK/cyc complexes have shown that plant Rb can be
readily phosphorylated by human CDK4/cycD,
CDK2/cycA and CDK2/cycE complexes [22 ••].
Moreover, an intact pocket domain is required for efficient phosphorylation by CDK4/cycD, as in human cells,
but not by the other complexes [22••]. Although the
functional significance of these studies still needs to be
addressed, it is conceivable that one (or more) plant
CDK complexed to cycD and/or cycA, functionally
equivalent to human CDK4/cycD and CDK2/cycA, regulates plant Rb function by sequential phosphorylation.
In this context, Rb has been also implicated in S phase
regulation in the absence of an intervening mitosis, as an
S-phase-associated kinase activity is increased in maize
endosperm, coinciding with the initiation of endoreduplication [12,23].
Figure 1
Comparison of plant and animal Rb family
members. The tree is based on amino acid
homologies of the region of each protein
containing the A/B pocket domain (black
boxes) and the spacer domain, using the
PILEUP program of the GCG package. Bars
at the right represent members of each
subgroup to show the overall conservation of
their domain organization. Note that the
sequences of RRB1 and ZmRb1 differ in
some residues, that the maize RRB2b has a
different carboxy-terminus and that Drosophila
RBF lacks the spacer domain. The accession
numbers of the sequences used in this study
are: AF007793, X98923, AF007795,
M26391, P33568, M15400, U00113,
A44879, Y09226, L14812, Q64701,
S67171, U50850 and X96975.
Maize RRB1
Maize ZmRb1
Maize RRB2b
Mouse
Rat
Human
Rb
Chicken
Xenopus
Newt
Human
p107
Mouse
Human
p130
Mouse
Drosophila RBF
A
B
Current Opinion in Plant Biology
494
Cell biology
Figure 2
Maize Rb
Human Rb
NEKCADVTIHIFFSKILKLAAIRIRNLCERVQCVEQ.TERVYNVFKQILEQQTT.LFFNRHIDQLILCCL
PLKSTSLSLFYKKVYRLAYLRLNTLCERLLSEHPELEHIIWTLFQHTLQNEYELMRDRHLDQIMMCSM
α11
α12
cyc fold α1
α13
α14
cyc fold α3
cyc fold α2
x
Tumor-derived mutations in human cells
x
YGVAKVCQLELTFREILNNYKREAQCKPEVFSSIYIGSTNRNGVLVSRHVGIITFYNEVFVPAAKPFLVSL
YGICKVKNIDLKFKIIVTAYKDLPHAVQETFKRVLI........KEEEYDSIIVFYNSVFMQRLKTNILQY
α15
cyc fold α4
The groove
LxCxE
binding
site
The rim
+
+
α16
+
+
β1
β2
α17
+
α18
cyc fold α5
+
Current Opinion in Plant Biology
Amino acid conservation and predicted structure of domain B of maize
Rb derived from the crystal structure of the A/B pocket of human Rb
[15]. Domain B contains eight α-helices (α11 through α18), five of
which participate in the so-called ‘cyclin fold’ (α-helices cycfold α1
through cycfold α5). The two short β strands (β1 and β2) are also
shown. Note that the eight amino acid insertion present in maize Rb is
predicted to be located in a loop between the β strands. Conserved
residues are shown in boldface: gray background, the LXCXE binding
groove (hydrophobic amino acids); black background, the rim
(positively charged amino acids), and empty boxes, the residues
frequently mutated in human tumors (X).
kinase (CAK) which is able to complement CAK mutations in yeasts and to specifically activate human CDK2
[24••]. Another plant gene with significant homology to
CAKs is the rice R2 gene whose expression is up-regulated in meristems and part of the elongation zone in
gibberellin-treated rice internodes, consistent with its
potential role in G1/S progression [25•]. On the other
side, the identification of other CDK-interacting proteins
such as a homolog of the yeast p13suc1 gene from
Arabidopsis [26] and an Arabidopsis CDK inhibitor (ICK1),
related to human p27 protein, which interacts with both
cdc2a and cycD3 and is induced by abscisic acid [27•]
should contribute significantly to our understanding of
the CDK regulatory network.
changes of the motif, for example LXCXK, drastically
reduce or abolish Rb-binding [13], whereas others, for
example LXCXQ, do not have such strong effects (L Liu
and J Stanley, personal communication). In Mastreviruses,
the Rep protein, required for viral DNA replication, is
translated from the same RepA-encoding mRNA after a
splicing event [29]. This Rep protein contains an LXCXE
motif but, interestingly, it does not bind to plant Rb ([28];
L Liu and J Stanley, personal communication; Q Xie and
C Gutierrez, unpublished data). Presumably, the LXCXE
motif is hidden since a carboxy-terminal deleted version of
WDV Rep can bind to plant Rb (Q Xie and C Gutierrez,
unpublished data). Second, the three Arabidopsis D-type
cyclins bind to maize Rb [22••]. Interaction between RRB1
and cycD3 in yeast seems to be enhanced in a carboxy-terminal deleted cycD3 protein [14•].
A broad spectrum of plant Rb-interacting
proteins
Since the isolation of cDNAs encoding plant Rb proteins, the identification of Rb-interacting proteins
(RbIPs) has allowed us to take a glimpse at the potential
roles of different Rb-containing complexes in plants.
RbIPs belong to two groups according to the domain
mediating binding to Rb.
LXCXE-containing RbIPs
First, as already mentioned, subgroup I geminiviruses
(Mastreviruses) encode a RepA protein that binds plant Rb
protein through its LXCXE motif. Examples are the wheat
dwarf virus (WDF; [13]), the maize streak virus (MSV;
[28]) or the bean yellow dwarf virus (BYDV; L Liu and
J Stanley, personal communication) RepA protein. Some
LXCXE-lacking RbIPs
Geminiviruses from the other two subgroups (II or
Curtoviruses and III or Begomoviruses), do not encode any
protein homologous to Mastrevirus RepA but they all encode
a Rep protein. Interestingly, Rep protein of tomato golden
mosaic virus, a Begomovirus, lacks an LXCXE motif but still
binds to maize RRB1 [14•]. Whether this represents a difference in the strategy used by different geminiviruses to
impinge on the Rb pathway is an open question. A second
set of proteins, related to the S. cerevisiae MSI1 protein [30]
and the human RbAp46 and RbAp48 proteins [31], has been
recently isolated from tomato (LeMSI1) and from
Arabidopsis (AtMSI1-3) [32•]. Their function is unknown,
but the human counterparts have been implicated in
The Rb pathway in plant cell cycle and development Gutierrez
processes related to chromatin remodelling [33,34].
Assuming similar roles in plants, it is conceivable that they
have implications in plant growth, morphogenesis and development. Third, a member of the E2F transcription factor
family has recently been cloned in plants [35•] and, like the
animal E2Fs, also lacks a LXCXE motif.
In addition, an increasing number of other proteins are
known to interact with Rb in human cells [36]. It is
expected, therefore, that more plant RBIPs will be identified in the near future. Among them, some could belong
to the family of human histone deacetylases which contribute to Rb-mediated transcriptional repression [37,38].
Others might be related to human oncogenes, differentiation and development factors. Still, it may occur that novel
RBIPs, unique to plants, may have evolved as a consequence of the specific demands of the plant body
organization and physiology.
the basal meristematic part and a progressive increase in differentiated cells along the leaf. In this differentiated zone,
Rb-immunoreactive ~110 kDa proteins accumulate, whereas
they are barely detected (or absent) in the basal zone [22••].
Plant E2F mRNA can be detected in proliferating as well as
in differentiated cells [35•]. These studies, together with the
increasing evidence of the role of cell proliferation, and perhaps cell cycle regulators, in plant development [46] suggest
a role for the components of the Rb pathway at some stages
during plant morphogenesis and patterning.
Conclusions and prospects
A summary of the current knowledge of the Rb pathway
in plants is shown in Figure 3. The picture is far from
Figure 3
Hormones/Nutrients
Regulation of G1/S-specific gene expression
by Rb-bound transcription factors
The correct passage of cells throughout the cell cycle
requires a strict temporal control of transcriptional activity
([3•] and references therein). For some genes, such as
those encoding ribonucleotide reductase, histones, and
cyclin D3, an up-regulation at G1/S and S phases has been
found [39–41]. The molecular nature of S-phase-specific
transcription factors, however, is not known. In human
cells, expression of G1/S genes frequently depends on
E2F transcription factors whose activity is modulated by
binding to Rb family members [5].
Auxin
Cytokinin
Sucrose
CDK
CycD3
CycD2
Geminiviruses
Rb - binding
proteins
The Rb pathway in plant differentiation and
development
Components of the Rb pathway are involved not only in cell
cycle control but also in mammalian [42,43] and Drosophila
development [44,45]. Rb also seems to play a role in growth
and development in plants. Thus, in maize leaves, a proliferative gradient exists with a high amount of dividing cells in
Negative
signals
CycD - CDK ?
CAK
?
P
CycD - CDK
?
CKI
E2F
DP ?
Rb
Is G1/S specific gene expression in plants mediated by
E2F-like proteins? The following evidence strongly suggests that this is the case: the ribonucleotide reductase and
cyclin D3 promoters, but not those of histone genes, contain binding sites similar to those of human E2F (C Gigot,
personal communication; J Murray, personal communication); the structural conservation of the A/B pocket
proteins [15]; maize Rb interacts, although weakly, with
human E2F-1 in yeast [14•,22••,35•]; and finally, maize Rb
represses E2F-dependent transcription in human cells
[22••]. As the identification of plant E2F-like protein(s)
and/or gene(s) has been so elusive to date, a significant
step ahead has been the recent isolation of a cDNA encoding a plant E2F [35•] which has a domain organization
similar that of the human E2F-1 subset of proteins. The
identification of this novel component of the Rb pathway
will allow further studies on its expression pattern and
transcriptional activity related to Rb binding.
495
Rb
P
E2F
Rb
DP ?
?
Differentiated
state
Current Opinion in Plant Biology
G1 / S
Transition
A proposal for the regulation of the Rb pathway. The key component of
the pathway is the complex constituted by Rb, E2F and DP proteins,
the latter still to be identified. Plant Rb contains several potential CDKphosphorylation sites (S/TP), in particular a carboxy-terminal cluster that
may be phosphorylated by CDK/cyc complexes, for example,
CDK/cycD. Changes in the phosphorylation state of Rb, and
presumably of its partners, could modulate their regulatory functions.
The release of the Rb-bound E2F/DP factors after Rb phosphorylation
by activated CDK/cycD would regulate genes required at the G1/S
transition. It should be kept in mind that up-regulation at G1/S may also
occur through other mechanisms. CDK is activated by specific
phosphorylation by CAKs and its function inhibited by specific inhibitors
(CKI). It is tempting to speculate that the expression of these G1/S
regulators and/or their activities may be regulated by hormones,
nutrients and/or negative growth signals. The normal Rb
phosphorylation pathway may be bypassed in geminivirus-infected cells
by the action of virally encoded Rb-binding proteins. The presence of
Rb and E2F messages in differentiated tissues suggests that, in
addition to their role during the cell cycle, Rb and E2F/DP complexes
may be crucial for the differentiation state of plant cells. These putative
interactions have not been demonstrated yet to occur in vivo.
496
Cell biology
complete, as many questions remain unanswered. On the
basis of recent results, however, the field is moving fast
and in extremely attractive directions. First, we need to
identify, clone and characterize at the molecular level
other components of the pathway. Second, since disruption of G1/S regulators in mammals leads to
transformation and tumor development, similar studies in
plants will serve to explain why they are extremely refractory to neoplastic transformation. Third, the normal G1/S
events seem to be altered by geminivirus infection as upregulation of genes, normally switched off in
differentiated cells, occurs. One possibility is that this
and/or other cellular effects of geminivirus proteins might
be a consequence of the interference with the Rb pathway. If this is the case, an abnormal S-phase, or at least
some S-phase functions and perhaps cellular DNA replication, could be triggered. Fourth, if it is proven that
Rb/E2F complexes participate in regulating differentiation decisions, a complete new window will be open to
look at plant development. These are just a few of many
aspects currently being investigated. Thus, we look forward to new and exciting results in the near future.
Acknowledgements
I thank D Dudits, C Gigot (deceased), H Hirt, D Inze, J Murray and J
Stanley for sharing ideas and communicating unpublished results, E
Martinez-Salas for comments on the manuscript, and past and present
members of the lab for contributing with their work, discussions and
stimulating environment. The work is partially supported by grants PB960919 (DGES), CI1*-CT94-0079 (European Union) and 06G/046/96
(Comunidad de Madrid) and by an institutional grant from Fundación
Ramón Areces.
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