Directory UMM :Data Elmu:jurnal:P:PlantScience:Plant Science_BioMedNet:061-080:
reviews
References
1
2
3
4
5
6
7
8
9
Neutra, M.R. et al. (1996) Antigen sampling across epithelial barriers
and induction of mucosal immune responses. Annu. Rev. Immunol. 14,
275–300
Jepson, M.A. and Clark, M.A. (1998) Studying M cells and their role in
infection. Trends Microbiol. 6, 359–365
Owen, R.L. (1999) Uptake and transport of intestinal macromolecules
and microorganisms by M cells in Peyer’s patches – a personal and
historical perspective. Semin. Immunol. 11, 157–163
Sansonetti, P.J. and Phalipon, A. (1999) M cells as ports of entry for
enteroinvasive pathogens: mechanisms of interaction, consequences for
the disease process. Semin. Immunol. 11, 193–203
Gebert, A. and Pabst, R. (1999) M cells at locations outside the gut.
Semin. Immunol. 11, 165–170
Freeman, T.C. et al. (1995) H+/di-tripeptide transporter (PepT1)
expression in the rabbit intestine. Pflügers Arch. – Eur. J. Physiol. 430,
394–400
Mason, C.M. et al. (1994) Heterogeneous Na+, K(+)-ATPase expression in
the epithelia of rabbit gut-associated lymphoid tissues. Pflügers Arch. –
Eur. J. Physiol. 427, 343–347
Smith, M.W. and Syme, G. (1982) Functional differentiation of
enterocytes in the follicle-associated epithelium of rat Peyer’s patch.
J. Cell Sci. 55, 147–156
Freeman, T.C. (1995) Parallel patterns of cell-specific gene expression
during enterocyte differentiation and maturation in the small intestine
of the rabbit. Differentiation 59, 179–192
Rho GTPases are small GTP-binding proteins of the
Ras superfamily. In animal cells, the Rho GTPase
family comprises three main subgroups, termed
Cdc42, Rac and, perhaps confusingly, Rho1.
Although the signalling outputs of the proteins
within these subgroups differ substantially, all share
certain fundamental characteristics. One is that
they act as molecular switches: on when bound to
GTP, off when this nucleotide is cleaved to GDP because of the intrinsic GTPase activity of the enzyme
or when no nucleotide is bound. A second is that
these switches are controlled by extracellular stimuli. A third shared property is that, in their activated
state, Rho proteins bind to a panoply of signalling
proteins that mediate the effects of these GTPases
on cell physiology2,3. These effects include a profound alteration in actin dynamics and intracellular
transport, activation of protein kinase cascades that
regulate gene transcription, cell-cycle transit and, in
some cell types, the assembly of the machinery
required for reactive oxidant production1. These
signalling properties of the Rho proteins are highly
conserved amongst eukaryotes as diverse as yeast,
slime molds, worms, flies and man, and, in many
cases, Rho proteins recruit and use very similar
effector proteins. For these reasons, one might predict that plant Rhos are regulated and function in a
manner similar to that seen in other eukaryotes, but,
as will be discussed, Rho signalling pathways in
plants take some unexpected turns.
Rho proteins: who’s who in plants?
In animal cells, the small GTPases of the Ras
superfamily can be divided into five families. These
are the Ras, Rab, Arf, Ran and Rho groups. Plants,
however, seem to lack one of these protein families
altogether, namely Ras. If Ras is indeed absent, this
puts plants into a category separate from most other
eukaryotes, in which Ras proteins play a central
trends in CELL BIOLOGY (Vol. 10) April 2000
10 Pappo, J. and Owen, R.L. (1988) Absence of secretory component
expression by epithelial cells overlying rabbit gut-associated lymphoid
tissue. Gastroenterology 95, 1173–1177
11 Brown, D. et al. (1990) Brush-border membrane alkaline phosphatase
activity in mouse Peyer’s patch follicle-associated enterocytes. J. Physiol.
427, 81–88
12 Smith, M.W. (1985) Selective expression of brush border hydrolases by
mouse Peyer’s patch and jejunal villus enterocytes. J. Cell. Physiol. 124,
219–225
13 Kelsall, B.L. and Strober, W. (1996) Distinct populations of dendritic
cells are present in the subepithelial dome and T cell regions of the
murine Peyer’s patch. J. Exp. Med. 183, 237–247
14 Neutra, M.R. (1999) M cells in antigen sampling of mucosal tissues.
Curr. Top. Microbiol. Immunol. 236, 17–32
15 Jepson, M.A. et al. (1992) Co-expression of vimentin and cytokeratins in
M cells of rabbit intestinal lymphoid follicle-associated epithelium.
Histochem. J. 24, 33–39
16 Gebert, A. et al. (1992) Co-localization of vimentin and cytokeratins in Mcells of rabbit gut-associated lymphoid tissue (GALT). Cell Tissue Res. 269,
331–340
17 Gebert, A. et al. (1994) Cytokeratin 18 is an M-cell marker in porcine
Peyer’s patches. Cell Tissue Res. 276, 213–221
18 Giannasca, P.J. et al. (1994) Regional differences in glycoconjugates of
intestinal M cells in mice: potential targets for mucosal vaccines. Am. J.
Physiol. 267, 1108–1121
19 Clark, M.A. et al. (1993) Differential expression of lectin-binding sites
Plant GTPases: the
Rhos in bloom
Aline H. Valster, Peter K. Hepler
and Jonathan Chernoff
In animal cells and in fungi, small GTP-binding proteins of the
Rho family have well-established roles in morphogenesis, cellcycle progression, gene transcription and the generation of
superoxide anions. The presence of these proteins in plant cells,
however, has been established only recently, and the role of Rho
GTPases in plants is now coming into view. Already, it is
apparent that there are both striking similarities and fascinating
differences in how Rho GTPases are regulated and used in plant
versus animal and fungal cells. These new findings define certain
core properties that might be common to members of this protein
family in all eukaryotes.
signalling role in growth and development, and
means that basic signalling pathways in organisms
in the plant kingdom might be organized according
to fundamentally different principles. In the case of
the Rho family, in plants there appears to be an overabundance of Rac-like proteins (in Arabidopsis and
0962-8924/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0962-8924(00)01728-1
141
reviews
TC10
pea, termed Rop, for ‘Rho of plants’, or Arac, for
‘Arabidopsis Rac’), but neither Cdc42s nor bona fide
Rhos
have yet been discovered (Fig. 1). Although the
Cdc42
Rac
Rops clearly belong to a distinct subfamily, they all
Sc
Cd
c4
bear the signature GTP-binding amino acid sequence
2
B
TKLD, a distinguishing mark of the Rac group, and
ac
R c3
m a
D R1
are therefore thought to be most closely related to
c
Ra c2
Ra
these GTPases (Fig. 2). Given that Rho itself has not
oA
Rh
been found in plants, it is therefore of considerable
Dm
A
Rho RhoB
interest that microinjection of a specific Rho
RhoC
Ara
inhibitor, C3 exotoxin, affects cytoplasmic streamc8
Arac10
ing in pea pollen tubes4 and in Tradescantia stamen
Sc Rh
o
hairs (see below). As the C3 toxin does not affect Racs
Sp Rho1
Rop6
(except under extraordinary in vitro conditions),
Rop2
4
Rop 3
Rop1 (A.H. Valster, unpublished) or members of the
p
Ro ac6
Rh
o6
Cdc42 family, these results suggest that either one of
Ar
the other (perhaps atypical) Rops is the in vivo C3
target, or this toxin is not as specific as currently beRop
lieved, or plants also encode authentic Rho proteins.
Rho
Two features stand out regarding Rop proteins in
plants: there are an extraordinary number of them
trends in Cell Biology
and they are extraordinarily alike. At the last count,
FIGURE 1
the Arabidopsis database contained at least 13 distinct
Rac-like genes, of which several potentially encode
Branches of the Rho family tree. The Rho family of GTPases comprises several distinct
subgroups, including Cdc42, Rac, Rho and Rop. In this diagram, phylogenetic
proteins that are nearly identical to one another5–11.
relationships are shown of representative Rho family proteins from plants, yeasts, flies
Multiple similar Rac-like genes have also been deand man. The Rop group is found only in plants and is most closely related by
scribed in other plants, representing both monocot
sequence to the Racs. The Rop and Arac proteins depicted here are from Arabidopsis,
and dicot species7,12–14. By contrast, only four Racs
but similar proteins have been found in many other plant species. Some branches of
are found in Caenorhabditis elegans, three, so far, in
the Rho subgroup tree have been foreshortened for clarity, as indicated by slash
humans, and none at all in budding or fission yeasts.
symbols. The phenogram was constructed using the Phylip programs. Dm, Drosophila
In Arabidopsis, the proteins encoded by the Rop2 and
melanogaster; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe.
Rop4 genes are 98% identical, whereas Rop1 and
Rop3 are 96% identical. The only major
GTP binding
Insert region
Effector loop
difference between these proteins and
several other closely related Rops/Aracs
NKFPSEYVPTVFDNYAVTVM
Cdc42
TQID
STIEKLAKNKQK
is found at their C termini, as is manifest
DQFPEVYVPTVFENYVADIE
RhoA
NKKD
HTRRELAKMKQE
by amino acid substitutions in polybasic
motifs similar to those that influence
Rac1
TKLD
DTIEKLKEKKLT
NAFPGEYIPTVFDNYSANVM
the subcellular localization of mammalian GTPases. These variations sugRop1
TKLD
QFFIDHPGAVPI
NTFPTDYVPTVFDNFSANVV
gest that Rops could be targeted to difRop2/Arac4
TKLD
QFFIDHPGAVPI
NTFPTDYVPTVFDNFSANVV
ferent locations. As a whole, the Rops
form a distinct and tightly clustered
Rop3/Arac1
TKLD
QFFIDHPGAVPI
NTFPTDYVPTVFDNFSANVV
branch of the Rho family tree, most
Rop4/Arac5
TKLD
QFFIDHPGAVPI
NTFPTDYVPTVFDNFSANVV
closely related (~70% similar) to the Rac
NTFPTDYVPTVFDNFSANIV
Rop6/Arac3
TKLD
QFFAEHPGAVPI
subfamily in animals. Only the putative
NTFPTDYVPTVFDNFSANVV
Arac6
TKLD
QFFIDHPGAVPI
proteins encoded by Arac7, Arac8 and
Arac10 show any truly distinguishing
Arac11
TKLD
QFFIDHPGAVPI
NTFPTDYVPTVFDNFSANVV
features, with marked clusters of amino
Arac2
TKLD
QFLKDHPGAASI
NTFPTDYVPTVFDNFSANVV
acid substitutions in the ‘insert region’,
Arac7
TKLD
GYLADHTNVITS
NKFPTDYIPTVFDNFSANVA
located between residues corresponding
Arac8
TKMD
HYLSDHPGLSPV
NKFPTDYIPTVFDNFSVNVV
to amino acids 124–135 in mammalian
Rac1 (Fig. 2). As this region is known to
Arac10
TKLD
HYLADHPGLSPV
NKFPTDYIPTVFDNFSVNVV
be crucial for the binding of specific
U88402
TKLD
LPDPSTGESDPV
NKFPEDYVPTVFENYTSKIT
effector proteins in mammalian and
trends in Cell Biology
fungal Rho-family proteins, it is likely
FIGURE 2 that these atypical GTPases have unique
signalling roles within the Rop family.
Rop subgroups. Cdc42, Rac and Rho proteins retain similar effector-binding domains but show
There is also at least one, much more
considerable variation in their GTP-binding motifs and in the insert region. In mammalian and fungal
distantly related, GTPase in Arabidopsis
Rho proteins, this latter region is important for determining the specificity of binding for some
(GenBank accession number U88402)
effector proteins. The plant Rops are most similar to the Rac group, both by overall homology and as
that encodes a Rop-related protein6.
seen from the effector-binding domain and the signature GTP-binding motif (yellow box). Although
This protein has both N- and C-terminal
the Rops are highly conserved, they can be subdivided into distinct groups based on differences in
the insert region, as shown in the green and blue shaded boxes.
insertions and is only ~50% identical to
Dm RacA
42
Dm Cdc
c42
Cd c42
Cd
Sp
7
ac
Ar
Sp
7
ho
R
o2
Rh
Arac2
Sp
Ro
p
Arac 1
11
c42
Cd
RhoG
142
trends in CELL BIOLOGY (Vol. 10) April 2000
reviews
the other Rop/Arac proteins. Most of the Rops and
Aracs have a C-terminal isoprenylation site, as is
common in Rho-family proteins in other organisms.
This modification means that Rops/Aracs are likely
to be present in membrane fractions in plant cells,
and indeed, in both pea and Arabidopsis, various
Rops localize to the plasma membrane in pollen
tubes11,15. In addition, in tobacco BY-2 cells, antisera
against human Rac1 detect immunoreactive material
in the membranes of intracellular organelles, possibly
representing Golgi stacks and peroxisomes16.
Why this profusion of Rops? In some cases, differential expression might be at play. Analysis of
expression patterns shows that, although many
Rops are expressed ubiquitously, Rop1 is expressed
only in mature pollen and pollen tubes, whereas
Arac2 is expressed exclusively in stems, roots and
hypocotyls. Nevertheless, the available sequence
and expression data favour the notion that at least
some of the Rops have redundant functions.
Perhaps this is one reason why genetic screens for
loss-of-function mutants in processes likely to involve
Rops have failed so far to yield genes encoding any
of these proteins.
Rop regulation: Dbl or nothing?
How are Rops regulated? Rho proteins, presumably
including plant Rops, are active when bound to GTP
and inactive when bound to GDP or when in a
nucleotide-free state. Rho GTPases are activated by
GDP–GTP exchange factors (GEFs) that contain
Dbl-homology (DH) domains. These proteins facilitate
the exchange of GDP for GTP in cells, thereby ‘loading’
the protein with GTP, resulting in conformational
changes and activation. There are two known classes
of negative regulators; Rho GTPase-activating proteins (Rho-GAPs) that increase the intrinsic GTPase
rate and thus inactivate Rho proteins, and guaninenucleotide dissociation inhibitors (Rho-GDIs) that
inhibit exchange of GDP for GTP and also block
translocation of Rho proteins to the plasma membrane. It is interesting to note that no Dbl-type GEF
sequences have yet been found in the Arabidopsis
database, even though the sequence of a substantial
percentage of the genome has been deduced. As the
bacterium Salmonella typhimurium has been shown
recently to encode a protein (SopE) that acts as a
GEF for Rac and Cdc42 but bears no sequence
homology whatsoever to Dbl proteins17, perhaps
plants activate Rops using a completely different,
and hence unrecognized, class of Rho-GEFs. As for
negative regulators, two-hybrid analysis of Ropbinding proteins from Lotus japonicus has uncovered
three putative GAP-like proteins that associate with
the Rops LjRac1 and LjRac218. Genes encoding similar proteins are found in the Arabidopsis database.
Importantly, these proteins have been shown to
possess actual GAP activity on Rops, indicating that
GAP proteins are likely to downregulate Rop function
in plant cells. Unlike any known mammalian or
yeast Rho-GAPs, these plant Rho-GAPs contain a
CRIB (Cdc42/Rac interactive binding) motif, an
element responsible for the binding of effector proteins to Rac and Cdc42 in animal cells19. Although
trends in CELL BIOLOGY (Vol. 10) April 2000
Rop
CLV receptor
PI-kinase
NADPH oxidase
MAPKs?
PtdIns(4,5)P2
H2O2
WUS inhibition
Ca2+
Cell polarity/
tip growth
Actin-binding Apoptosis
proteins
Cellulose
synthesis
Actin
cytoskeleton
Secondary
wall formation
Pathogen
defence
Meristem
signalling
trends in Cell Biology
FIGURE 3
Rop-regulated pathways. Rop activity has been correlated with the processes
indicated in the square boxes. Parts of the signal-transduction pathways that have
been elucidated to date are indicated. Arrows do not necessarily indicate direct
interactions, and question mark indicates hypothetical aspect of this model. Crosstalk
between pathways is expected to occur, as in, for example, the cell polarity and the
actin cytoskeleton pathways. Also, a single Rop-regulated process, such as the
production of reactive oxygen species, might affect both pathogen defence and
secondary wall formation. CLV receptor, CLAVATA receptor; MAPKs, mitogenactivated protein kinases; PI, phosphatidylinositol; PtdIns(4,5)P2, phosphatidylinositol
(4,5)-bisphosphate; WUS, WUSCHEL protein.
the requirement for the CRIB motif for binding or
regulating Rops has not yet been demonstrated formally, it is tempting to speculate that, unlike animal
or fungal Rho-GAPs, plant Rho-GAPs target their
substrates (i.e. Rops) through a CRIB domain.
Indeed, as all known CRIB proteins bind only to the
activated forms of GTPases, such a mechanism
could ensure that Rop signals are terminated efficiently. It is also possible that these plant Rho-GAPs
play effector roles, as has been proposed for Ras
GAPs in mammalian cells20,21. As for other potential
Rop regulators, two putative Rho-GDIs are found in
the Arabidopsis sequence database, although these
have not yet been shown to regulate Rops. Thus, of
the three well-recognized mechanisms for Rho
regulation (i.e. GEFs, GAPs and GDIs), in plants only
inhibition by GAPs and probably GDIs can be
inferred at present, and the overall regulation of
Rops therefore remains something of an enigma.
Rop function: Rac redux?
Mammalian and fungal GTPases are well known
for their ability to regulate cell morphology. Plant
Rops appear to have retained this basic function.
The importance of the Rop proteins in cell polarity
and tip growth has been best established in pollen
tubes. Pollen tubes are single cells that are characterized by the presence of strong polarity. Localized
secretion and a steep intracellular Ca2+ gradient at
the tip of the pollen tube facilitate tip growth22–24,
allowing the cell to extend in order to bridge the
considerable gap between stigma and ovule – a
Aline Valster and
Peter Hepler are in
the Biology Dept,
University of
Massachusetts,
Morrill Science
Center III,
Amherst, MA
01003, USA; and
Jonathan Chernoff
is in the Molecular
Oncology
Program, Fox
Chase Cancer
Center, 7701
Burholme Ave,
Philadelphia, PA
19111, USA.
E-mails: avalster@
bio.umass.edu;
hepler@
bio.umass.edu;
j_chernoff@
fccc.edu
143
reviews
(a)
(c)
(b)
growth, although tip integrity is maintained4. This
effect by the dominant–negative Rop mutant is
surprising, as such mutants are thought to act by
sequestering endogenous Dbl-type GEFs25, of which
plants apparently have none. Although the identity
of the effectors of Rops are not yet known, there are
indications that, like Rac in animal cells, Rops
associate with and regulate phosphatidylinositol
monophosphate (PtdInsP) kinase activity11. As Rop1
is localized to the pollen tube tip, the resulting increase in phosphatidylinositol (4,5)-bisphosphate
[PtdIns(4,5)P2] might well be involved in the regulation of secretion and/or actin organization
through its effects on the Ca2+ gradient and on
actin-binding proteins such as gelsolin, villin and
profilin. It is particularly interesting that an increase
in extracellular Ca2+ partly suppresses4, whereas a
decrease enhances26, the effects of loss of Rop1 function on pollen tube growth. These findings suggest
that Rop1 and Ca2+ might act in a common signalling pathway that affects cell polarity; a connection that has not yet been reported for Rho proteins
in other organisms (Fig. 3).
(d)
trends in Cell Biology
FIGURE 4
Effects of a Rho inhibitor on plant cell architecture. The Rho-specific toxin C3
(1 mg ml–1) was injected into interphase Tradescantia stamen hair cells. (a)
Differential interference contrast image of a cell before injection, showing
transvacuolar strands and cytoplasmic streaming; (b) same cell 15 minutes after C3
injection, transvacuolar strands have broken down and cytoplasmic streaming has
ceased; (c) confocal image of the same C3-injected cell, stained with fluorescein
isothiocyanate (FITC)-phalloidin, revealing breakdown of cytoplasmic actin filaments.
The cortical actin array (out of focal plane) is intact; (d) control cell, stained with
FITC-phalloidin, showing actin filaments in the cytoplasmic strands. Bar, 10 mm.
distance that can span several centimetres.
Arabidopsis Rop1 and the nearly identical Arac6
(also known as At-Rac2) protein are pollen-specific
Rops that are strongly localized to the plasma membrane at the apical domain of the pollen tube, suggesting a role in establishing cell polarity and tip
growth11,15. Indeed, in Arabidopsis, overexpression
of a constitutively active form of this protein results
in depolarized growth and isodiametric swelling of
the pollen tube tip4,15. Interestingly, expression of
activated plant Rop1 in fission yeast also induces
isotropic cell growth, indicating that a conserved set
of effectors might be used by evolutionarily disparate members of the Rho family9. In pollen tubes,
overexpression of a dominant–negative mutant of
Rop1, in addition to microinjection of inhibitory
antibodies against Rop1, results in inhibition of tip
144
Acting on actin?
So, we know that Rops affect plant cell morphology
and polarity, but how do they do it? One of the bestestablished functions of the Rho proteins in animal
and yeast cells is their involvement in regulating the
actin cytoskeleton. Expression of activated forms of
Rho, Rac and Cdc42 proteins results in distinctive
cellular and cytoskeletal phenotypes, in large part
due to changes in the rate, character and location of
actin polymerization and organization27. In plant
cells, however, the evidence that Rho-like proteins
are involved in regulation of the actin cytoskeleton
is quite limited. Overexpression of a constitutively
active mutant of Rop in tobacco pollen tubes results
in an aberrant actin organization in the swollen
pollen tube tip11. In normal pollen tubes, actin bundles are organized parallel to the length of the tube,
whereas tobacco pollen tubes transfected with activated Rop develop excessively thick actin bundles
coiled in a helical pattern11. In addition, microinjection of the Rho-inhibitor C3 exotoxin has been
shown to interfere with cytoplasmic streaming, a
well-established actin-based process, in pea pollen
tube4. In interphase Tradescantia stamen hair cells,
C3 toxin causes cytoplasmic strands to break down
and inhibits cytoplasmic streaming (Fig. 4).
However, as noted previously, the C3 findings are
intriguing but also peculiar, as Rho itself has not yet
been found in plants and C3 does not inhibit Rop1.
These results suggest that plant Rops, like their
counterparts in other organisms, regulate actin dynamics. What is missing is detailed knowledge of
Rop effectors that could connect this GTPase to
proteins that are involved more directly in regulating
actin structure in plants. PtdInsP kinase could represent one such effector, but, based on what we
know about GTPases in other systems, it seems unlikely to be the only one. Biochemical purifications,
two-hybrid screens and, perhaps, genetic analyses
will undoubtedly help to fill in this incomplete
trends in CELL BIOLOGY (Vol. 10) April 2000
reviews
picture. As mentioned previously, two-hybrid
screens for Rop-binding partners in Lotus18 and
Arabidopsis (Z. Yang, pers. commun.) have already
revealed that proteins containing CRIB motifs are
present in plants. In animal cells, several CRIB-containing proteins, such as p21-activated kinases28,29,
Wiskott–Aldrich protein30 and MSE55/Borg531,32,
regulate actin dynamics. Whether equivalents for
these proteins will be found in plants, however,
remains to be seen.
A role in defending the home turf?
Another physiological role for Rop has been established in the regulation of the elicitor-induced
oxidative burst that accompanies host–pathogen
interactions. This burst results in the production of
reactive oxygen species (ROS) that can trigger the
death of infected host cells through apoptosis33,34.
Like the oxidative burst in mammalian neutrophils,
ROS in plants can be generated by an NADPH oxidase
enzyme complex. In neutrophils, ROS production is
governed by Rac1 and Rac2, which are required for assembly of the multicomponent oxidase complex that
includes several so-called ‘phox’ proteins35. Although
the composition of the plant NADPH oxidase complex
is not as well understood as its animal counterpart, it
is known to contain p47-phox and p67-phox36, and
homologues of the essential gp91-phox subunit have
also been found recently37–39. In addition, in tobacco
cells, a Rac homologue has been identified immunologically as a component of the NADPH enzyme complex associated with ROS production40. Evidence that
plant Racs regulate ROS production and cell death
derives from a series of experiments in which constitutively active and dominant–negative mutants of
OsRac1 (a Rac-like protein from rice that is most similar to Arabidopsis Arac7) were introduced into rice14. The
constitutively active OsRac1 induced ROS production
and cell death with apoptotic characteristics, whereas
the dominant–negative OsRac1 prevented ROS production and apoptosis in cells treated with the protein
phosphatase inhibitor calyculin A. These results mean
that certain Rops/Aracs, possibly through recruitment
and assembly of an oxidase complex, are likely to be
involved in pathogen defence in plants (Fig. 3).
…and in building stronger walls?
A recent report suggests another role for Rac-like
proteins and ROS production in development and
cell differentiation. Here, a Rac-like protein from
cotton is hypothesized to be involved in the transition
of primary to secondary cell wall formation. In cotton fibre formation, a model system for the study of
secondary wall formation, Potikha et al. have established that an increase in H2O2 production accompanies the transition from primary to secondary
wall formation, and that this molecule might act as
a messenger in the process of inducing secondary
wall formation41. Coincident with deposition of the
secondary wall and production of the H2O2, Rac13 (a
protein 90% identical to Arabidopsis Arac2) is highly
expressed12. Indeed, when Arabidopsis or soy bean
cell cultures are transformed with the constitutively
active form of the cotton Rac13, H2O2 production is
trends in CELL BIOLOGY (Vol. 10) April 2000
stimulated, whereas transformation of the dominant–
negative form of Rac13 or with antisense constructs
results in decreased H2O2 levels41. These observations, together with the connection between Rops
and ROS formation in pathogen defence, indicate
that Rac13 is a regulator of H2O2 production and,
through this mechanism, could stimulate secondary
cell wall formation and/or cellulose synthesis.
Putting Rops in their place
A role for Rop in plant morphology and development has been proposed recently. A receptor-like
kinase (RLK) in Arabidopsis, termed CLAVATA1
(CLV1), has been identified that is involved in
meristem signalling42. This signalling pathway
regulates the balance between cell multiplication
and differentiation in the meristem. Clv1 mutant
plants show enlarged meristems (with up to a
1000-fold increase in undifferentiated cells) owing
to a lack of cell differentiation that normally follows
cell division. The CLV1 RLK is a transmembrane
receptor with an extracellular leucine-rich repeat
domain and an intracellular serine/threonine
kinase domain. Using co-immunoprecipitation,
Trotochaud et al.43 established that the CLV1
protein forms a large signalling complex that includes
the CLV3 protein, KAPP (a kinase-associated protein
phosphatase) and a Rop. The signalling complex is
detected in two distinct forms of 185 kDa and
450 kDa. Rop is associated exclusively with the
450-kDa complex that is thought to represent the
active form of the signalling complex. Although this
Rop has not been shown to be regulated through its
association with CLV1, nor to bind directly to this
RLK, it is interesting to contemplate the possibility
that Rop acts immediately downstream of a cell-surface receptor and, possibly, upstream of a mitogenactivated protein kinase (MAPK) cascade44.
Tantalizing, but preliminary, support for the latter
aspect of this model is provided by evidence from
two-hybrid screens that show that certain as-yetuncharacterized Raf-like kinases can associate with
LjRac119. In turn, the MAPK pathway is thought to
inactivate the WUSCHEL gene that is involved in
normal maintenance of meristems45.
In mammalian systems, Rho GTPases can be activated by G-protein-coupled receptors and by receptor protein tyrosine kinases. In the case of G-proteincoupled receptors, the route to some Rho GTPases
might be relatively direct as two groups have reported that a Dbl-type GEF for RhoA binds directly
to the alpha subunit of a heterotrimeric G protein46,47.
The connection between receptor protein tyrosine
kinases and Rho proteins, however, is thought to be
indirect, involving multiple layers of adaptor and
regulatory proteins, and no firm connection has
been established between receptor serine/threonine
kinases and Rho proteins. Therefore, the observation of a possible direct link between a serine/
threonine kinase cell-surface signalling complex
and a small GTPase in the Arabidopsis meristem signaltransduction system suggests that plants have
adopted a unique strategy to regulate the function
of small GTPases.
145
reviews
What can we expect to dig up next?
The sheer abundance and extreme conservation of
the Rops argue for a crucial role in plant physiology.
It is now clear that these proteins are distant
cousins, rather than near siblings, of the more
familiar Rho/Rac/Cdc42 groups in other eukaryotes
and that they therefore could be subject to different
control mechanisms and might have adopted
unique signalling roles. That Rops might be regulated in a novel manner can be inferred from their
coprecipitation with an RLK complex and also by
the surprising absence (so far) in plant databases of
sequences encoding the expected key regulatory
proteins, such as classical Rho-GEFs. Furthermore,
one group of plausible regulatory proteins we do
know in plants (i.e. the GAPs) has apparently
hijacked a targeting motif (the CRIB domain) and
used it in a manner never seen before in any other
system. The core signalling activities of this protein
family – regulation of cell morphology, ROS production and, perhaps, gene transcription – do
appear to be retained in plant Rops. However, nothing
is known of their effector proteins. This situation is
likely to be remedied soon as plant scientists gear-up
their Rop pull-down assays and two-hybrid screens.
Thus, we can expect to see soon just how differently
the plant kingdom has decided to employ these
small but powerful signalling proteins.
References
1
2
3
4
5
6
7
8
9
10
11
12
Acknowledgements
We thank our
many colleagues
who furnished
information in
advance of
publication, and
Roland Dunbrack
for his assistance
in the
construction of
dendrograms.
146
13
14
15
16
Mackay, D.J.G. and Hall, A. (1998) Rho GTPases. J. Biol. Chem. 273,
20685–20688
Aspenstrom, P. (1999) Effectors for the Rho GTPases. Curr. Opin. Cell
Biol. 11, 95–102
Van Aelst, L. and D’Souza-Schorey, C. (1997) Rho GTPases and
signalling networks. Genes Dev. 11, 2295–2322
Lin, Y. and Yang, Z. (1997) Inhibition of pollen tube elongation by
microinjected anti-Rop1Ps antibodies suggests a crucial role for Rhotype GTPases in the control of tip growth. Plant Cell 9, 1647–1659
Xia, G. et al. (1996) Identification of a plant cytoskeletal, cell cyclerelated and polarity-related proteins using Schizosaccharomyces pombe.
Plant J. 10, 761–769
Collins, C.C. and Johnson, D.I. (1997) An Arabidopsis thaliana expressed
sequence tag cDNA that encodes a Rac-like protein. Plant Physiol. 113, 1463
Bischoff, F. et al. (1999) GTP-binding proteins in plants. Cell. Mol. Life
Sci. 55, 233–256
Winge, P. et al. (1997) Cloning and characterization of rac-like cDNAs
from Arabidopsis thaliana. Plant Mol. Biol. 35, 483–495
Li, H. et al. (1998) Arabidopsis Rho-related GTPases: differential gene
expression in pollen and polar localization in fission yeast. Plant Physiol.
18, 407–417
Yang, Z. and Watson, J.C. (1993) Molecular cloning and
characterization of rho, a ras-related small GTP-binding protein from
garden pea. Proc. Natl. Acad. Sci. U. S. A. 90, 8732–8736
Kost, B. et al. (1999) Rac homologues and compartmentalized
phosphatidylinositol 4,5-bisphosphate act in a common pathway to
regulate polar pollen tube growth. J. Cell Biol. 145, 317–330
Delmer, D.P. et al. (1995) Genes encoding small GTP-binding proteins
analogous to mammalian rac are preferentially expressed in developing
cotton fibers. Mol. Gen. Genet. 248, 43–51
Borg, S. et al. (1997) Identification of new protein species among 33
different small GTP-binding proteins encoded by cDNAs from Lotus
japonicus, and expression of corresponding mRNAs in developing root
nodules. Plant J. 11, 237–250
Kawasaki, T. et al. (1999) The small GTP-binding protein Rac is a
regulator of cell death in plants. Proc. Natl. Acad. Sci. U. S. A. 96,
10922–10926
Lin, Y. et al. (1996) Localization of a Rho GTPase implies a role in tip
growth and movement of the generative cell in pollen tubes. Plant Cell
8, 293–303
Couchy, I. et al. (1998) Immunodetection of Rho-like plant proteins
with Rac1 and Cdc42Hs antibodies. J. Exp. Bot. 49, 1647–1659
17 Hardt, W.D. et al. (1998) S. typhimurium encodes an activator of Rho
GTPases that induces membrane ruffling and nuclear responses in host
cells. Cell 93, 815–826
18 Borg, S. et al. (1999) Plant cell growth and differentiation may involve
GAP regulation of Rac activity. FEBS Lett. 453, 341–345
19 Burbelo, P.D. et al. (1995) A conserved binding motif defines numerous
candidate target proteins for both Cdc42 and Rac GTPases. J. Biol.
Chem. 270, 29071–29074
20 Bollag, G. and McCormick, F. (1992) GTPase activating proteins. Semin.
Cancer Biol. 3, 199–208
21 Tocque, B. et al. (1997) Ras-GTPase activating protein (GAP); a putative
effector for Ras. Cell. Signal. 9, 153–158
22 Pierson, E.S. et al. (1994) Pollen tubes growth is coupled to the
extracellular calcium ion flux and the intracellular calcium gradient:
effect of BAPTA-type buffers and hypertonic media. Plant Cell 6,
1815–1828
23 Holdaway-Clarke, T.L. et al. (1997) Pollen tube growth and the
intracellular calcium gradient oscillate in phase while extracellular
calcium influx is delayed. Plant Cell 9, 1999–2010
24 Yang, Z. (1998) Signalling tip growth in plants. Curr. Opin. Plant Biol. 1,
525–530
25 Feig, L.A. (1999) Tools of the trade: use of dominant-inhibitory mutants
of Ras-family GTPases. Nat. Cell Biol. 1, 25–27
26 Li, H. et al. (1999) Control of pollen tube tip growth by a Rop GTPasedependent pathway that leads to tip-localized calcium influx. Plant Cell
11, 1731–1742
27 Hall, A. (1998) Rho GTPases and the actin cytoskeleton. Science 279,
509–514
28 Sells, M.A. and Chernoff, J. (1997) Emerging from the Pak: the p21activated protein kinase family. Trends Cell Biol. 7, 162–167
29 Bagrodia, S. and Cerione, R.A. (1999) PAK to the future. Trends Cell Biol.
9, 350–355
30 Symons, M. et al. (1996) Wiskott–Aldrich syndrome protein, a novel
effector for the GTPase CDC42Hs, is implicated in actin polymerization.
Cell 84, 723–734
31 Burbelo, P.D. et al. (1999) MSE55, a Cdc42 effector protein, induces
long cellular extensions in fibroblasts. Proc. Natl. Acad. Sci. U. S. A. 96,
9083–9088
32 Joberty, G. et al. (1999) The Borgs, a new family of Cdc42 and TC10
GTPase-interacting proteins. Mol. Cell. Biol. 19, 6585–6597
33 Levine, A. et al. (1994) H2O2 from the oxidative burst orchestrates the
plant hypersensitive disease resistance response. Cell 79, 583–593
34 Mehdy, M.C. et al. (1996) The role of activated oxygen species in plant
disease resistance. Physiol. Plant. 98, 365–374
35 Bokoch, G.M. (1995) Regulation of phagocyte respiratory burst by small
GTP-binding proteins. Trends Cell Biol. 5, 109–113
36 Dwyer, S.C. et al. (1996) Plant and human neutrophil oxidative burst
complexes contain immunologically related proteins. Biochim. Biophys.
Acta 1289, 231–237
37 Groom, Q.J. et al. (1996) RbohA, a rice homolog of the mammalian
gp91 phox respiratory burst oxidase gene. Plant J. 10, 515–522
38 Keller, T. et al. (1998) A plant homolog of the neutrophil NADPH
oxidase gp91phox subunit gene encodes a plasma membrane protein
with Ca2+ binding motifs. Plant Cell 10, 255–266
39 Torres, M.A. et al. (1998) Six Arabidopsis thaliana homologues of the
human respiratory burst oxidase (gp91phox). Plant J. 14, 365–370
40 Kieffer, F. et al. (1997) Tobacco cells contain a protein, immunologically
related to the neutrophil small G protein Rac2 and involved in elicitorinduced oxidative burst. FEBS Lett. 403, 149–153
41 Potikha, T. et al. (1999) The involvement of hydrogen peroxide in the
differentiation of secondary walls in cotton fibers. Plant Physiol. 119,
849–858
42 Clark, S.E. et al. (1997) The CLAVATA1 gene encodes a putative receptor
kinase that controls shoot and floral meristem size in Arabidopsis. Cell
89, 575–585
43 Trotochaud, A.E. et al. (1999) The CLAVATA1 receptor-like
kinase requires CLAVATA3 for its assembly into a signalling
complex that includes KAPP and a Rho-related protein. Plant Cell 11,
393–406
44 Kohorn, B.D. (1999) Shuffling the deck: plant signalling plays a club.
Trends Cell Biol. 9, 381–383
45 Laux, T. et al. (1996) The WUSCHEL gene is required for shoot and floral
meristem integrity in Arabidopsis. Development 122, 87–96
46 Hart, M.J. et al. (1998) Direct stimulation of the guanine nucleotide
exchange activity of p115 RhoGEF by Galpha13. Science 280,
2112–2114
47 Kozasa, T. et al. (1998) p115 RhoGEF, a GTPase activating protein for
Galpha12 and Galpha13. Science 280, 2109–2111
trends in CELL BIOLOGY (Vol. 10) April 2000
References
1
2
3
4
5
6
7
8
9
Neutra, M.R. et al. (1996) Antigen sampling across epithelial barriers
and induction of mucosal immune responses. Annu. Rev. Immunol. 14,
275–300
Jepson, M.A. and Clark, M.A. (1998) Studying M cells and their role in
infection. Trends Microbiol. 6, 359–365
Owen, R.L. (1999) Uptake and transport of intestinal macromolecules
and microorganisms by M cells in Peyer’s patches – a personal and
historical perspective. Semin. Immunol. 11, 157–163
Sansonetti, P.J. and Phalipon, A. (1999) M cells as ports of entry for
enteroinvasive pathogens: mechanisms of interaction, consequences for
the disease process. Semin. Immunol. 11, 193–203
Gebert, A. and Pabst, R. (1999) M cells at locations outside the gut.
Semin. Immunol. 11, 165–170
Freeman, T.C. et al. (1995) H+/di-tripeptide transporter (PepT1)
expression in the rabbit intestine. Pflügers Arch. – Eur. J. Physiol. 430,
394–400
Mason, C.M. et al. (1994) Heterogeneous Na+, K(+)-ATPase expression in
the epithelia of rabbit gut-associated lymphoid tissues. Pflügers Arch. –
Eur. J. Physiol. 427, 343–347
Smith, M.W. and Syme, G. (1982) Functional differentiation of
enterocytes in the follicle-associated epithelium of rat Peyer’s patch.
J. Cell Sci. 55, 147–156
Freeman, T.C. (1995) Parallel patterns of cell-specific gene expression
during enterocyte differentiation and maturation in the small intestine
of the rabbit. Differentiation 59, 179–192
Rho GTPases are small GTP-binding proteins of the
Ras superfamily. In animal cells, the Rho GTPase
family comprises three main subgroups, termed
Cdc42, Rac and, perhaps confusingly, Rho1.
Although the signalling outputs of the proteins
within these subgroups differ substantially, all share
certain fundamental characteristics. One is that
they act as molecular switches: on when bound to
GTP, off when this nucleotide is cleaved to GDP because of the intrinsic GTPase activity of the enzyme
or when no nucleotide is bound. A second is that
these switches are controlled by extracellular stimuli. A third shared property is that, in their activated
state, Rho proteins bind to a panoply of signalling
proteins that mediate the effects of these GTPases
on cell physiology2,3. These effects include a profound alteration in actin dynamics and intracellular
transport, activation of protein kinase cascades that
regulate gene transcription, cell-cycle transit and, in
some cell types, the assembly of the machinery
required for reactive oxidant production1. These
signalling properties of the Rho proteins are highly
conserved amongst eukaryotes as diverse as yeast,
slime molds, worms, flies and man, and, in many
cases, Rho proteins recruit and use very similar
effector proteins. For these reasons, one might predict that plant Rhos are regulated and function in a
manner similar to that seen in other eukaryotes, but,
as will be discussed, Rho signalling pathways in
plants take some unexpected turns.
Rho proteins: who’s who in plants?
In animal cells, the small GTPases of the Ras
superfamily can be divided into five families. These
are the Ras, Rab, Arf, Ran and Rho groups. Plants,
however, seem to lack one of these protein families
altogether, namely Ras. If Ras is indeed absent, this
puts plants into a category separate from most other
eukaryotes, in which Ras proteins play a central
trends in CELL BIOLOGY (Vol. 10) April 2000
10 Pappo, J. and Owen, R.L. (1988) Absence of secretory component
expression by epithelial cells overlying rabbit gut-associated lymphoid
tissue. Gastroenterology 95, 1173–1177
11 Brown, D. et al. (1990) Brush-border membrane alkaline phosphatase
activity in mouse Peyer’s patch follicle-associated enterocytes. J. Physiol.
427, 81–88
12 Smith, M.W. (1985) Selective expression of brush border hydrolases by
mouse Peyer’s patch and jejunal villus enterocytes. J. Cell. Physiol. 124,
219–225
13 Kelsall, B.L. and Strober, W. (1996) Distinct populations of dendritic
cells are present in the subepithelial dome and T cell regions of the
murine Peyer’s patch. J. Exp. Med. 183, 237–247
14 Neutra, M.R. (1999) M cells in antigen sampling of mucosal tissues.
Curr. Top. Microbiol. Immunol. 236, 17–32
15 Jepson, M.A. et al. (1992) Co-expression of vimentin and cytokeratins in
M cells of rabbit intestinal lymphoid follicle-associated epithelium.
Histochem. J. 24, 33–39
16 Gebert, A. et al. (1992) Co-localization of vimentin and cytokeratins in Mcells of rabbit gut-associated lymphoid tissue (GALT). Cell Tissue Res. 269,
331–340
17 Gebert, A. et al. (1994) Cytokeratin 18 is an M-cell marker in porcine
Peyer’s patches. Cell Tissue Res. 276, 213–221
18 Giannasca, P.J. et al. (1994) Regional differences in glycoconjugates of
intestinal M cells in mice: potential targets for mucosal vaccines. Am. J.
Physiol. 267, 1108–1121
19 Clark, M.A. et al. (1993) Differential expression of lectin-binding sites
Plant GTPases: the
Rhos in bloom
Aline H. Valster, Peter K. Hepler
and Jonathan Chernoff
In animal cells and in fungi, small GTP-binding proteins of the
Rho family have well-established roles in morphogenesis, cellcycle progression, gene transcription and the generation of
superoxide anions. The presence of these proteins in plant cells,
however, has been established only recently, and the role of Rho
GTPases in plants is now coming into view. Already, it is
apparent that there are both striking similarities and fascinating
differences in how Rho GTPases are regulated and used in plant
versus animal and fungal cells. These new findings define certain
core properties that might be common to members of this protein
family in all eukaryotes.
signalling role in growth and development, and
means that basic signalling pathways in organisms
in the plant kingdom might be organized according
to fundamentally different principles. In the case of
the Rho family, in plants there appears to be an overabundance of Rac-like proteins (in Arabidopsis and
0962-8924/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0962-8924(00)01728-1
141
reviews
TC10
pea, termed Rop, for ‘Rho of plants’, or Arac, for
‘Arabidopsis Rac’), but neither Cdc42s nor bona fide
Rhos
have yet been discovered (Fig. 1). Although the
Cdc42
Rac
Rops clearly belong to a distinct subfamily, they all
Sc
Cd
c4
bear the signature GTP-binding amino acid sequence
2
B
TKLD, a distinguishing mark of the Rac group, and
ac
R c3
m a
D R1
are therefore thought to be most closely related to
c
Ra c2
Ra
these GTPases (Fig. 2). Given that Rho itself has not
oA
Rh
been found in plants, it is therefore of considerable
Dm
A
Rho RhoB
interest that microinjection of a specific Rho
RhoC
Ara
inhibitor, C3 exotoxin, affects cytoplasmic streamc8
Arac10
ing in pea pollen tubes4 and in Tradescantia stamen
Sc Rh
o
hairs (see below). As the C3 toxin does not affect Racs
Sp Rho1
Rop6
(except under extraordinary in vitro conditions),
Rop2
4
Rop 3
Rop1 (A.H. Valster, unpublished) or members of the
p
Ro ac6
Rh
o6
Cdc42 family, these results suggest that either one of
Ar
the other (perhaps atypical) Rops is the in vivo C3
target, or this toxin is not as specific as currently beRop
lieved, or plants also encode authentic Rho proteins.
Rho
Two features stand out regarding Rop proteins in
plants: there are an extraordinary number of them
trends in Cell Biology
and they are extraordinarily alike. At the last count,
FIGURE 1
the Arabidopsis database contained at least 13 distinct
Rac-like genes, of which several potentially encode
Branches of the Rho family tree. The Rho family of GTPases comprises several distinct
subgroups, including Cdc42, Rac, Rho and Rop. In this diagram, phylogenetic
proteins that are nearly identical to one another5–11.
relationships are shown of representative Rho family proteins from plants, yeasts, flies
Multiple similar Rac-like genes have also been deand man. The Rop group is found only in plants and is most closely related by
scribed in other plants, representing both monocot
sequence to the Racs. The Rop and Arac proteins depicted here are from Arabidopsis,
and dicot species7,12–14. By contrast, only four Racs
but similar proteins have been found in many other plant species. Some branches of
are found in Caenorhabditis elegans, three, so far, in
the Rho subgroup tree have been foreshortened for clarity, as indicated by slash
humans, and none at all in budding or fission yeasts.
symbols. The phenogram was constructed using the Phylip programs. Dm, Drosophila
In Arabidopsis, the proteins encoded by the Rop2 and
melanogaster; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe.
Rop4 genes are 98% identical, whereas Rop1 and
Rop3 are 96% identical. The only major
GTP binding
Insert region
Effector loop
difference between these proteins and
several other closely related Rops/Aracs
NKFPSEYVPTVFDNYAVTVM
Cdc42
TQID
STIEKLAKNKQK
is found at their C termini, as is manifest
DQFPEVYVPTVFENYVADIE
RhoA
NKKD
HTRRELAKMKQE
by amino acid substitutions in polybasic
motifs similar to those that influence
Rac1
TKLD
DTIEKLKEKKLT
NAFPGEYIPTVFDNYSANVM
the subcellular localization of mammalian GTPases. These variations sugRop1
TKLD
QFFIDHPGAVPI
NTFPTDYVPTVFDNFSANVV
gest that Rops could be targeted to difRop2/Arac4
TKLD
QFFIDHPGAVPI
NTFPTDYVPTVFDNFSANVV
ferent locations. As a whole, the Rops
form a distinct and tightly clustered
Rop3/Arac1
TKLD
QFFIDHPGAVPI
NTFPTDYVPTVFDNFSANVV
branch of the Rho family tree, most
Rop4/Arac5
TKLD
QFFIDHPGAVPI
NTFPTDYVPTVFDNFSANVV
closely related (~70% similar) to the Rac
NTFPTDYVPTVFDNFSANIV
Rop6/Arac3
TKLD
QFFAEHPGAVPI
subfamily in animals. Only the putative
NTFPTDYVPTVFDNFSANVV
Arac6
TKLD
QFFIDHPGAVPI
proteins encoded by Arac7, Arac8 and
Arac10 show any truly distinguishing
Arac11
TKLD
QFFIDHPGAVPI
NTFPTDYVPTVFDNFSANVV
features, with marked clusters of amino
Arac2
TKLD
QFLKDHPGAASI
NTFPTDYVPTVFDNFSANVV
acid substitutions in the ‘insert region’,
Arac7
TKLD
GYLADHTNVITS
NKFPTDYIPTVFDNFSANVA
located between residues corresponding
Arac8
TKMD
HYLSDHPGLSPV
NKFPTDYIPTVFDNFSVNVV
to amino acids 124–135 in mammalian
Rac1 (Fig. 2). As this region is known to
Arac10
TKLD
HYLADHPGLSPV
NKFPTDYIPTVFDNFSVNVV
be crucial for the binding of specific
U88402
TKLD
LPDPSTGESDPV
NKFPEDYVPTVFENYTSKIT
effector proteins in mammalian and
trends in Cell Biology
fungal Rho-family proteins, it is likely
FIGURE 2 that these atypical GTPases have unique
signalling roles within the Rop family.
Rop subgroups. Cdc42, Rac and Rho proteins retain similar effector-binding domains but show
There is also at least one, much more
considerable variation in their GTP-binding motifs and in the insert region. In mammalian and fungal
distantly related, GTPase in Arabidopsis
Rho proteins, this latter region is important for determining the specificity of binding for some
(GenBank accession number U88402)
effector proteins. The plant Rops are most similar to the Rac group, both by overall homology and as
that encodes a Rop-related protein6.
seen from the effector-binding domain and the signature GTP-binding motif (yellow box). Although
This protein has both N- and C-terminal
the Rops are highly conserved, they can be subdivided into distinct groups based on differences in
the insert region, as shown in the green and blue shaded boxes.
insertions and is only ~50% identical to
Dm RacA
42
Dm Cdc
c42
Cd c42
Cd
Sp
7
ac
Ar
Sp
7
ho
R
o2
Rh
Arac2
Sp
Ro
p
Arac 1
11
c42
Cd
RhoG
142
trends in CELL BIOLOGY (Vol. 10) April 2000
reviews
the other Rop/Arac proteins. Most of the Rops and
Aracs have a C-terminal isoprenylation site, as is
common in Rho-family proteins in other organisms.
This modification means that Rops/Aracs are likely
to be present in membrane fractions in plant cells,
and indeed, in both pea and Arabidopsis, various
Rops localize to the plasma membrane in pollen
tubes11,15. In addition, in tobacco BY-2 cells, antisera
against human Rac1 detect immunoreactive material
in the membranes of intracellular organelles, possibly
representing Golgi stacks and peroxisomes16.
Why this profusion of Rops? In some cases, differential expression might be at play. Analysis of
expression patterns shows that, although many
Rops are expressed ubiquitously, Rop1 is expressed
only in mature pollen and pollen tubes, whereas
Arac2 is expressed exclusively in stems, roots and
hypocotyls. Nevertheless, the available sequence
and expression data favour the notion that at least
some of the Rops have redundant functions.
Perhaps this is one reason why genetic screens for
loss-of-function mutants in processes likely to involve
Rops have failed so far to yield genes encoding any
of these proteins.
Rop regulation: Dbl or nothing?
How are Rops regulated? Rho proteins, presumably
including plant Rops, are active when bound to GTP
and inactive when bound to GDP or when in a
nucleotide-free state. Rho GTPases are activated by
GDP–GTP exchange factors (GEFs) that contain
Dbl-homology (DH) domains. These proteins facilitate
the exchange of GDP for GTP in cells, thereby ‘loading’
the protein with GTP, resulting in conformational
changes and activation. There are two known classes
of negative regulators; Rho GTPase-activating proteins (Rho-GAPs) that increase the intrinsic GTPase
rate and thus inactivate Rho proteins, and guaninenucleotide dissociation inhibitors (Rho-GDIs) that
inhibit exchange of GDP for GTP and also block
translocation of Rho proteins to the plasma membrane. It is interesting to note that no Dbl-type GEF
sequences have yet been found in the Arabidopsis
database, even though the sequence of a substantial
percentage of the genome has been deduced. As the
bacterium Salmonella typhimurium has been shown
recently to encode a protein (SopE) that acts as a
GEF for Rac and Cdc42 but bears no sequence
homology whatsoever to Dbl proteins17, perhaps
plants activate Rops using a completely different,
and hence unrecognized, class of Rho-GEFs. As for
negative regulators, two-hybrid analysis of Ropbinding proteins from Lotus japonicus has uncovered
three putative GAP-like proteins that associate with
the Rops LjRac1 and LjRac218. Genes encoding similar proteins are found in the Arabidopsis database.
Importantly, these proteins have been shown to
possess actual GAP activity on Rops, indicating that
GAP proteins are likely to downregulate Rop function
in plant cells. Unlike any known mammalian or
yeast Rho-GAPs, these plant Rho-GAPs contain a
CRIB (Cdc42/Rac interactive binding) motif, an
element responsible for the binding of effector proteins to Rac and Cdc42 in animal cells19. Although
trends in CELL BIOLOGY (Vol. 10) April 2000
Rop
CLV receptor
PI-kinase
NADPH oxidase
MAPKs?
PtdIns(4,5)P2
H2O2
WUS inhibition
Ca2+
Cell polarity/
tip growth
Actin-binding Apoptosis
proteins
Cellulose
synthesis
Actin
cytoskeleton
Secondary
wall formation
Pathogen
defence
Meristem
signalling
trends in Cell Biology
FIGURE 3
Rop-regulated pathways. Rop activity has been correlated with the processes
indicated in the square boxes. Parts of the signal-transduction pathways that have
been elucidated to date are indicated. Arrows do not necessarily indicate direct
interactions, and question mark indicates hypothetical aspect of this model. Crosstalk
between pathways is expected to occur, as in, for example, the cell polarity and the
actin cytoskeleton pathways. Also, a single Rop-regulated process, such as the
production of reactive oxygen species, might affect both pathogen defence and
secondary wall formation. CLV receptor, CLAVATA receptor; MAPKs, mitogenactivated protein kinases; PI, phosphatidylinositol; PtdIns(4,5)P2, phosphatidylinositol
(4,5)-bisphosphate; WUS, WUSCHEL protein.
the requirement for the CRIB motif for binding or
regulating Rops has not yet been demonstrated formally, it is tempting to speculate that, unlike animal
or fungal Rho-GAPs, plant Rho-GAPs target their
substrates (i.e. Rops) through a CRIB domain.
Indeed, as all known CRIB proteins bind only to the
activated forms of GTPases, such a mechanism
could ensure that Rop signals are terminated efficiently. It is also possible that these plant Rho-GAPs
play effector roles, as has been proposed for Ras
GAPs in mammalian cells20,21. As for other potential
Rop regulators, two putative Rho-GDIs are found in
the Arabidopsis sequence database, although these
have not yet been shown to regulate Rops. Thus, of
the three well-recognized mechanisms for Rho
regulation (i.e. GEFs, GAPs and GDIs), in plants only
inhibition by GAPs and probably GDIs can be
inferred at present, and the overall regulation of
Rops therefore remains something of an enigma.
Rop function: Rac redux?
Mammalian and fungal GTPases are well known
for their ability to regulate cell morphology. Plant
Rops appear to have retained this basic function.
The importance of the Rop proteins in cell polarity
and tip growth has been best established in pollen
tubes. Pollen tubes are single cells that are characterized by the presence of strong polarity. Localized
secretion and a steep intracellular Ca2+ gradient at
the tip of the pollen tube facilitate tip growth22–24,
allowing the cell to extend in order to bridge the
considerable gap between stigma and ovule – a
Aline Valster and
Peter Hepler are in
the Biology Dept,
University of
Massachusetts,
Morrill Science
Center III,
Amherst, MA
01003, USA; and
Jonathan Chernoff
is in the Molecular
Oncology
Program, Fox
Chase Cancer
Center, 7701
Burholme Ave,
Philadelphia, PA
19111, USA.
E-mails: avalster@
bio.umass.edu;
hepler@
bio.umass.edu;
j_chernoff@
fccc.edu
143
reviews
(a)
(c)
(b)
growth, although tip integrity is maintained4. This
effect by the dominant–negative Rop mutant is
surprising, as such mutants are thought to act by
sequestering endogenous Dbl-type GEFs25, of which
plants apparently have none. Although the identity
of the effectors of Rops are not yet known, there are
indications that, like Rac in animal cells, Rops
associate with and regulate phosphatidylinositol
monophosphate (PtdInsP) kinase activity11. As Rop1
is localized to the pollen tube tip, the resulting increase in phosphatidylinositol (4,5)-bisphosphate
[PtdIns(4,5)P2] might well be involved in the regulation of secretion and/or actin organization
through its effects on the Ca2+ gradient and on
actin-binding proteins such as gelsolin, villin and
profilin. It is particularly interesting that an increase
in extracellular Ca2+ partly suppresses4, whereas a
decrease enhances26, the effects of loss of Rop1 function on pollen tube growth. These findings suggest
that Rop1 and Ca2+ might act in a common signalling pathway that affects cell polarity; a connection that has not yet been reported for Rho proteins
in other organisms (Fig. 3).
(d)
trends in Cell Biology
FIGURE 4
Effects of a Rho inhibitor on plant cell architecture. The Rho-specific toxin C3
(1 mg ml–1) was injected into interphase Tradescantia stamen hair cells. (a)
Differential interference contrast image of a cell before injection, showing
transvacuolar strands and cytoplasmic streaming; (b) same cell 15 minutes after C3
injection, transvacuolar strands have broken down and cytoplasmic streaming has
ceased; (c) confocal image of the same C3-injected cell, stained with fluorescein
isothiocyanate (FITC)-phalloidin, revealing breakdown of cytoplasmic actin filaments.
The cortical actin array (out of focal plane) is intact; (d) control cell, stained with
FITC-phalloidin, showing actin filaments in the cytoplasmic strands. Bar, 10 mm.
distance that can span several centimetres.
Arabidopsis Rop1 and the nearly identical Arac6
(also known as At-Rac2) protein are pollen-specific
Rops that are strongly localized to the plasma membrane at the apical domain of the pollen tube, suggesting a role in establishing cell polarity and tip
growth11,15. Indeed, in Arabidopsis, overexpression
of a constitutively active form of this protein results
in depolarized growth and isodiametric swelling of
the pollen tube tip4,15. Interestingly, expression of
activated plant Rop1 in fission yeast also induces
isotropic cell growth, indicating that a conserved set
of effectors might be used by evolutionarily disparate members of the Rho family9. In pollen tubes,
overexpression of a dominant–negative mutant of
Rop1, in addition to microinjection of inhibitory
antibodies against Rop1, results in inhibition of tip
144
Acting on actin?
So, we know that Rops affect plant cell morphology
and polarity, but how do they do it? One of the bestestablished functions of the Rho proteins in animal
and yeast cells is their involvement in regulating the
actin cytoskeleton. Expression of activated forms of
Rho, Rac and Cdc42 proteins results in distinctive
cellular and cytoskeletal phenotypes, in large part
due to changes in the rate, character and location of
actin polymerization and organization27. In plant
cells, however, the evidence that Rho-like proteins
are involved in regulation of the actin cytoskeleton
is quite limited. Overexpression of a constitutively
active mutant of Rop in tobacco pollen tubes results
in an aberrant actin organization in the swollen
pollen tube tip11. In normal pollen tubes, actin bundles are organized parallel to the length of the tube,
whereas tobacco pollen tubes transfected with activated Rop develop excessively thick actin bundles
coiled in a helical pattern11. In addition, microinjection of the Rho-inhibitor C3 exotoxin has been
shown to interfere with cytoplasmic streaming, a
well-established actin-based process, in pea pollen
tube4. In interphase Tradescantia stamen hair cells,
C3 toxin causes cytoplasmic strands to break down
and inhibits cytoplasmic streaming (Fig. 4).
However, as noted previously, the C3 findings are
intriguing but also peculiar, as Rho itself has not yet
been found in plants and C3 does not inhibit Rop1.
These results suggest that plant Rops, like their
counterparts in other organisms, regulate actin dynamics. What is missing is detailed knowledge of
Rop effectors that could connect this GTPase to
proteins that are involved more directly in regulating
actin structure in plants. PtdInsP kinase could represent one such effector, but, based on what we
know about GTPases in other systems, it seems unlikely to be the only one. Biochemical purifications,
two-hybrid screens and, perhaps, genetic analyses
will undoubtedly help to fill in this incomplete
trends in CELL BIOLOGY (Vol. 10) April 2000
reviews
picture. As mentioned previously, two-hybrid
screens for Rop-binding partners in Lotus18 and
Arabidopsis (Z. Yang, pers. commun.) have already
revealed that proteins containing CRIB motifs are
present in plants. In animal cells, several CRIB-containing proteins, such as p21-activated kinases28,29,
Wiskott–Aldrich protein30 and MSE55/Borg531,32,
regulate actin dynamics. Whether equivalents for
these proteins will be found in plants, however,
remains to be seen.
A role in defending the home turf?
Another physiological role for Rop has been established in the regulation of the elicitor-induced
oxidative burst that accompanies host–pathogen
interactions. This burst results in the production of
reactive oxygen species (ROS) that can trigger the
death of infected host cells through apoptosis33,34.
Like the oxidative burst in mammalian neutrophils,
ROS in plants can be generated by an NADPH oxidase
enzyme complex. In neutrophils, ROS production is
governed by Rac1 and Rac2, which are required for assembly of the multicomponent oxidase complex that
includes several so-called ‘phox’ proteins35. Although
the composition of the plant NADPH oxidase complex
is not as well understood as its animal counterpart, it
is known to contain p47-phox and p67-phox36, and
homologues of the essential gp91-phox subunit have
also been found recently37–39. In addition, in tobacco
cells, a Rac homologue has been identified immunologically as a component of the NADPH enzyme complex associated with ROS production40. Evidence that
plant Racs regulate ROS production and cell death
derives from a series of experiments in which constitutively active and dominant–negative mutants of
OsRac1 (a Rac-like protein from rice that is most similar to Arabidopsis Arac7) were introduced into rice14. The
constitutively active OsRac1 induced ROS production
and cell death with apoptotic characteristics, whereas
the dominant–negative OsRac1 prevented ROS production and apoptosis in cells treated with the protein
phosphatase inhibitor calyculin A. These results mean
that certain Rops/Aracs, possibly through recruitment
and assembly of an oxidase complex, are likely to be
involved in pathogen defence in plants (Fig. 3).
…and in building stronger walls?
A recent report suggests another role for Rac-like
proteins and ROS production in development and
cell differentiation. Here, a Rac-like protein from
cotton is hypothesized to be involved in the transition
of primary to secondary cell wall formation. In cotton fibre formation, a model system for the study of
secondary wall formation, Potikha et al. have established that an increase in H2O2 production accompanies the transition from primary to secondary
wall formation, and that this molecule might act as
a messenger in the process of inducing secondary
wall formation41. Coincident with deposition of the
secondary wall and production of the H2O2, Rac13 (a
protein 90% identical to Arabidopsis Arac2) is highly
expressed12. Indeed, when Arabidopsis or soy bean
cell cultures are transformed with the constitutively
active form of the cotton Rac13, H2O2 production is
trends in CELL BIOLOGY (Vol. 10) April 2000
stimulated, whereas transformation of the dominant–
negative form of Rac13 or with antisense constructs
results in decreased H2O2 levels41. These observations, together with the connection between Rops
and ROS formation in pathogen defence, indicate
that Rac13 is a regulator of H2O2 production and,
through this mechanism, could stimulate secondary
cell wall formation and/or cellulose synthesis.
Putting Rops in their place
A role for Rop in plant morphology and development has been proposed recently. A receptor-like
kinase (RLK) in Arabidopsis, termed CLAVATA1
(CLV1), has been identified that is involved in
meristem signalling42. This signalling pathway
regulates the balance between cell multiplication
and differentiation in the meristem. Clv1 mutant
plants show enlarged meristems (with up to a
1000-fold increase in undifferentiated cells) owing
to a lack of cell differentiation that normally follows
cell division. The CLV1 RLK is a transmembrane
receptor with an extracellular leucine-rich repeat
domain and an intracellular serine/threonine
kinase domain. Using co-immunoprecipitation,
Trotochaud et al.43 established that the CLV1
protein forms a large signalling complex that includes
the CLV3 protein, KAPP (a kinase-associated protein
phosphatase) and a Rop. The signalling complex is
detected in two distinct forms of 185 kDa and
450 kDa. Rop is associated exclusively with the
450-kDa complex that is thought to represent the
active form of the signalling complex. Although this
Rop has not been shown to be regulated through its
association with CLV1, nor to bind directly to this
RLK, it is interesting to contemplate the possibility
that Rop acts immediately downstream of a cell-surface receptor and, possibly, upstream of a mitogenactivated protein kinase (MAPK) cascade44.
Tantalizing, but preliminary, support for the latter
aspect of this model is provided by evidence from
two-hybrid screens that show that certain as-yetuncharacterized Raf-like kinases can associate with
LjRac119. In turn, the MAPK pathway is thought to
inactivate the WUSCHEL gene that is involved in
normal maintenance of meristems45.
In mammalian systems, Rho GTPases can be activated by G-protein-coupled receptors and by receptor protein tyrosine kinases. In the case of G-proteincoupled receptors, the route to some Rho GTPases
might be relatively direct as two groups have reported that a Dbl-type GEF for RhoA binds directly
to the alpha subunit of a heterotrimeric G protein46,47.
The connection between receptor protein tyrosine
kinases and Rho proteins, however, is thought to be
indirect, involving multiple layers of adaptor and
regulatory proteins, and no firm connection has
been established between receptor serine/threonine
kinases and Rho proteins. Therefore, the observation of a possible direct link between a serine/
threonine kinase cell-surface signalling complex
and a small GTPase in the Arabidopsis meristem signaltransduction system suggests that plants have
adopted a unique strategy to regulate the function
of small GTPases.
145
reviews
What can we expect to dig up next?
The sheer abundance and extreme conservation of
the Rops argue for a crucial role in plant physiology.
It is now clear that these proteins are distant
cousins, rather than near siblings, of the more
familiar Rho/Rac/Cdc42 groups in other eukaryotes
and that they therefore could be subject to different
control mechanisms and might have adopted
unique signalling roles. That Rops might be regulated in a novel manner can be inferred from their
coprecipitation with an RLK complex and also by
the surprising absence (so far) in plant databases of
sequences encoding the expected key regulatory
proteins, such as classical Rho-GEFs. Furthermore,
one group of plausible regulatory proteins we do
know in plants (i.e. the GAPs) has apparently
hijacked a targeting motif (the CRIB domain) and
used it in a manner never seen before in any other
system. The core signalling activities of this protein
family – regulation of cell morphology, ROS production and, perhaps, gene transcription – do
appear to be retained in plant Rops. However, nothing
is known of their effector proteins. This situation is
likely to be remedied soon as plant scientists gear-up
their Rop pull-down assays and two-hybrid screens.
Thus, we can expect to see soon just how differently
the plant kingdom has decided to employ these
small but powerful signalling proteins.
References
1
2
3
4
5
6
7
8
9
10
11
12
Acknowledgements
We thank our
many colleagues
who furnished
information in
advance of
publication, and
Roland Dunbrack
for his assistance
in the
construction of
dendrograms.
146
13
14
15
16
Mackay, D.J.G. and Hall, A. (1998) Rho GTPases. J. Biol. Chem. 273,
20685–20688
Aspenstrom, P. (1999) Effectors for the Rho GTPases. Curr. Opin. Cell
Biol. 11, 95–102
Van Aelst, L. and D’Souza-Schorey, C. (1997) Rho GTPases and
signalling networks. Genes Dev. 11, 2295–2322
Lin, Y. and Yang, Z. (1997) Inhibition of pollen tube elongation by
microinjected anti-Rop1Ps antibodies suggests a crucial role for Rhotype GTPases in the control of tip growth. Plant Cell 9, 1647–1659
Xia, G. et al. (1996) Identification of a plant cytoskeletal, cell cyclerelated and polarity-related proteins using Schizosaccharomyces pombe.
Plant J. 10, 761–769
Collins, C.C. and Johnson, D.I. (1997) An Arabidopsis thaliana expressed
sequence tag cDNA that encodes a Rac-like protein. Plant Physiol. 113, 1463
Bischoff, F. et al. (1999) GTP-binding proteins in plants. Cell. Mol. Life
Sci. 55, 233–256
Winge, P. et al. (1997) Cloning and characterization of rac-like cDNAs
from Arabidopsis thaliana. Plant Mol. Biol. 35, 483–495
Li, H. et al. (1998) Arabidopsis Rho-related GTPases: differential gene
expression in pollen and polar localization in fission yeast. Plant Physiol.
18, 407–417
Yang, Z. and Watson, J.C. (1993) Molecular cloning and
characterization of rho, a ras-related small GTP-binding protein from
garden pea. Proc. Natl. Acad. Sci. U. S. A. 90, 8732–8736
Kost, B. et al. (1999) Rac homologues and compartmentalized
phosphatidylinositol 4,5-bisphosphate act in a common pathway to
regulate polar pollen tube growth. J. Cell Biol. 145, 317–330
Delmer, D.P. et al. (1995) Genes encoding small GTP-binding proteins
analogous to mammalian rac are preferentially expressed in developing
cotton fibers. Mol. Gen. Genet. 248, 43–51
Borg, S. et al. (1997) Identification of new protein species among 33
different small GTP-binding proteins encoded by cDNAs from Lotus
japonicus, and expression of corresponding mRNAs in developing root
nodules. Plant J. 11, 237–250
Kawasaki, T. et al. (1999) The small GTP-binding protein Rac is a
regulator of cell death in plants. Proc. Natl. Acad. Sci. U. S. A. 96,
10922–10926
Lin, Y. et al. (1996) Localization of a Rho GTPase implies a role in tip
growth and movement of the generative cell in pollen tubes. Plant Cell
8, 293–303
Couchy, I. et al. (1998) Immunodetection of Rho-like plant proteins
with Rac1 and Cdc42Hs antibodies. J. Exp. Bot. 49, 1647–1659
17 Hardt, W.D. et al. (1998) S. typhimurium encodes an activator of Rho
GTPases that induces membrane ruffling and nuclear responses in host
cells. Cell 93, 815–826
18 Borg, S. et al. (1999) Plant cell growth and differentiation may involve
GAP regulation of Rac activity. FEBS Lett. 453, 341–345
19 Burbelo, P.D. et al. (1995) A conserved binding motif defines numerous
candidate target proteins for both Cdc42 and Rac GTPases. J. Biol.
Chem. 270, 29071–29074
20 Bollag, G. and McCormick, F. (1992) GTPase activating proteins. Semin.
Cancer Biol. 3, 199–208
21 Tocque, B. et al. (1997) Ras-GTPase activating protein (GAP); a putative
effector for Ras. Cell. Signal. 9, 153–158
22 Pierson, E.S. et al. (1994) Pollen tubes growth is coupled to the
extracellular calcium ion flux and the intracellular calcium gradient:
effect of BAPTA-type buffers and hypertonic media. Plant Cell 6,
1815–1828
23 Holdaway-Clarke, T.L. et al. (1997) Pollen tube growth and the
intracellular calcium gradient oscillate in phase while extracellular
calcium influx is delayed. Plant Cell 9, 1999–2010
24 Yang, Z. (1998) Signalling tip growth in plants. Curr. Opin. Plant Biol. 1,
525–530
25 Feig, L.A. (1999) Tools of the trade: use of dominant-inhibitory mutants
of Ras-family GTPases. Nat. Cell Biol. 1, 25–27
26 Li, H. et al. (1999) Control of pollen tube tip growth by a Rop GTPasedependent pathway that leads to tip-localized calcium influx. Plant Cell
11, 1731–1742
27 Hall, A. (1998) Rho GTPases and the actin cytoskeleton. Science 279,
509–514
28 Sells, M.A. and Chernoff, J. (1997) Emerging from the Pak: the p21activated protein kinase family. Trends Cell Biol. 7, 162–167
29 Bagrodia, S. and Cerione, R.A. (1999) PAK to the future. Trends Cell Biol.
9, 350–355
30 Symons, M. et al. (1996) Wiskott–Aldrich syndrome protein, a novel
effector for the GTPase CDC42Hs, is implicated in actin polymerization.
Cell 84, 723–734
31 Burbelo, P.D. et al. (1999) MSE55, a Cdc42 effector protein, induces
long cellular extensions in fibroblasts. Proc. Natl. Acad. Sci. U. S. A. 96,
9083–9088
32 Joberty, G. et al. (1999) The Borgs, a new family of Cdc42 and TC10
GTPase-interacting proteins. Mol. Cell. Biol. 19, 6585–6597
33 Levine, A. et al. (1994) H2O2 from the oxidative burst orchestrates the
plant hypersensitive disease resistance response. Cell 79, 583–593
34 Mehdy, M.C. et al. (1996) The role of activated oxygen species in plant
disease resistance. Physiol. Plant. 98, 365–374
35 Bokoch, G.M. (1995) Regulation of phagocyte respiratory burst by small
GTP-binding proteins. Trends Cell Biol. 5, 109–113
36 Dwyer, S.C. et al. (1996) Plant and human neutrophil oxidative burst
complexes contain immunologically related proteins. Biochim. Biophys.
Acta 1289, 231–237
37 Groom, Q.J. et al. (1996) RbohA, a rice homolog of the mammalian
gp91 phox respiratory burst oxidase gene. Plant J. 10, 515–522
38 Keller, T. et al. (1998) A plant homolog of the neutrophil NADPH
oxidase gp91phox subunit gene encodes a plasma membrane protein
with Ca2+ binding motifs. Plant Cell 10, 255–266
39 Torres, M.A. et al. (1998) Six Arabidopsis thaliana homologues of the
human respiratory burst oxidase (gp91phox). Plant J. 14, 365–370
40 Kieffer, F. et al. (1997) Tobacco cells contain a protein, immunologically
related to the neutrophil small G protein Rac2 and involved in elicitorinduced oxidative burst. FEBS Lett. 403, 149–153
41 Potikha, T. et al. (1999) The involvement of hydrogen peroxide in the
differentiation of secondary walls in cotton fibers. Plant Physiol. 119,
849–858
42 Clark, S.E. et al. (1997) The CLAVATA1 gene encodes a putative receptor
kinase that controls shoot and floral meristem size in Arabidopsis. Cell
89, 575–585
43 Trotochaud, A.E. et al. (1999) The CLAVATA1 receptor-like
kinase requires CLAVATA3 for its assembly into a signalling
complex that includes KAPP and a Rho-related protein. Plant Cell 11,
393–406
44 Kohorn, B.D. (1999) Shuffling the deck: plant signalling plays a club.
Trends Cell Biol. 9, 381–383
45 Laux, T. et al. (1996) The WUSCHEL gene is required for shoot and floral
meristem integrity in Arabidopsis. Development 122, 87–96
46 Hart, M.J. et al. (1998) Direct stimulation of the guanine nucleotide
exchange activity of p115 RhoGEF by Galpha13. Science 280,
2112–2114
47 Kozasa, T. et al. (1998) p115 RhoGEF, a GTPase activating protein for
Galpha12 and Galpha13. Science 280, 2109–2111
trends in CELL BIOLOGY (Vol. 10) April 2000