During the development of multicellular organisms

many cell types are produced. Dependi ng on thei r

position, each cell perceives different signals, responds
through intracellular signalling pathways and, even-
tually, adopts a specific cell fate. Subsequent cell dif-
ferenti ati on usually i nvolves complex changes. For
example, cells exit the mitotic cycle or enter cycles of
ENDOREDUPLICATION, the cellular archi tecture alters to
meet the functi onal requi rements of the respective
cell type, and the metaboli sm of the cell changes
accordi ng to i ts functi on. Compared wi th ani mals,
plant development faces addi ti onal constrai nts
because the ri gi d cell walls prevent any cell move-
ment. In plants, a few si ngle-celled Arabidopsis
thaliana model systems i n parti cular root hai rs
and trichomes have greatly improved our under-
standing of the development of single cells.
The goal of this review is to summarize how the
study of A. thalianatrichomes facilitates the under-
standing of development at the single-cell level. A large
number of mutants have been characterized that
enabled the identification of subsequent developmental
processes. These include the selection of trichomes in a
field of epidermal cells, cell-fate determination, changes
in the cell-cycle mode and cell-shape control. The
genetic, molecular and cell-biological analysis of tri-
chome development has revealed only a few trichome-
specific processes, as most developmental steps involve
the regulation of general cellular machineries. Therefore,
studying the trichome system has provided unique
insights into the function of transcription factors, the
microtubule and actin cytoskeleton, the cell cycle and
cell-death control. The study of all the developmental
stages of a single cell is a first step towards an under-
standing of how general cellular processes are inte-
grated during development.
Steps in trichome development
Shoot epidermal hairs are known as trichomes, a term
that is derived from the Greek word for hairs, trichos.
Trichomes are found in most plants and can comprise
either single or several cells and can be secretory glan-
dular or nonglandular
1,2
. The functions that are
ascribed to trichomes range from protecting the plant
against insect herbivores and UV light, to reducing
transpiration and increasing tolerance to freezing
3,4
.
Trichomes are an excellent model system because
they are of epidermal origin and are therefore easily
accessible. In addition, A. thalianatrichomes are not
essential for the plant under laboratory conditions,
which facilitates the isolation of trichome-specific
mutants
5,6
. So far, most studies have been carried out
on leaf trichomes (FIG. 1). At the base of young leaves,
single cells that are spaced out at regular distances in an
area of apparently equivalent PROTODERMAL CELLSdevelop
into trichomes
7,8
. Incipient trichomes stop mitotic cell
divisions and initiate endoreduplication cycles. As a
result, the trichome cell increases in size and changes its
direction of growth such that it grows perpendicular to
the leaf surface. Further growth is characterized by a
total of about four endoreduplication cycles that result
in a DNA content of 32C (1C is the DNA content of
PLANT TRICHOMES: A MODEL FOR
CELL DIFFERENTIATION
Martin Hlskamp
D uring the past few years, the focus in plant developm ental biology has shifted from studying the
organization of the w hole body or individual organs tow ards the behaviour of the sm allest unit of
the organism , the single cell. Plant leaf hairs, or trichom es, serve as an excellent m odel system to
study all aspects of plant differentiation at the single-cell level, including the choice of cell fate,
developm ental control of the cell cycle, cell polarity and the control of cell shape.
NATURE REVI EWS | MOLECULAR CELL BIOLOGY VOLUME 5 |JUNE 2004 | 4 7 1
University of Kln,Botanical
InstituteIII,Gyrhofstrae15,
50931 Kln,Germany.
e-mail: martin.huelskamp@
uni-koeln.de
doi:10.1038/nrm1404
ENDOREDUPLICATION
A modified cell cyclein which
DNA replication continuesin
theabsenceof mitosisand
cytokinesis.
PROTODERMAL CELL
A young epidermal cell that has
not yet differentiated into a
specialized cell type.
R E V I E WS
PL ANT CE L L BI OLOGY
2004 Nature PublishingGroup
4 7 2 | JUNE 2004 |VOLUME 5 www.nature.com/reviews/molcellbio
R E V I E WS
emerging picture is that only very few genes are, in fact,
trichome specific. Most genes are relevant for many cell
typesand are involved in general cellular processes(FIG. 1).
It seems that mutations in these genes have little effect in
most cell types but are crucial during trichome develop-
ment possibly because trichomes, with their rapid
growth and enormoussize,are more demanding.
Trichome patterning and initiation
Wild-type trichomes are initiated with an average dis-
tance of about three cells between developing trichomes,
and almost never form directly next to each other as
would be expected if they were randomly distributed
which indicates that there must be an underlying pat-
terning mechani sm
7,8
. A mechani sm that would
explain trichome patterning by a standardized cell-
division pattern that segregates trichome cell fates was
excluded by clonal analysis
8,11
. It is therefore hypothe-
si zed that tri chome selecti on i s based on a mutual-
inhibition mechanism
1215
(FIG. 2): cells that are initially
equivalent produce a trichome-promoting factor (or
factors) that activates a factor (or factors) that sup-
presses trichome development in the neighbouring cells.
This way, cells begin to compete and, due to stochastic
the unreplicated haploid genome), which is accompa-
nied by rapid cell enlargement
7,9
. The growing cell
undergoes two consecutive branching events, the orien-
tations of which are co-aligned with respect to the
basaldistal leaf axis
10
.
A large number of mutations that affect trichome
development were identified in several mutagenesis
screens
5,6
. The mutations helped to define regulatory
processes in trichome development according to their
specific developmental defects(FIG.1).The selection of tri-
chome cellsand the initiation of the trichome cell fate are
under the control of a small group of so-called patterning
genes. One gene seems to specifically translate the pat-
terning cuesinto cell-fate differentiation.The switch from
mitotic cycles to endoreduplication cycles and the num-
ber of endoreduplication cycles are controlled by the
endoreduplication genes. A large number of genes are
known to be important for branching. The directionality
of expansion growth is affected in the so-called distorted
mutants.One mutant isknown to cause unscheduled cell
death, and several other mutantsseem to affect the matu-
ration of the trichome.
Now that the genetic interactions are well under-
stood and most of the genes have been cloned, the
Figure 1 |Trichome development.A schem atic presentation of trichom e developm ent is show n at the top. B elow , representative
exam ples of m utant or overexpression phenotypes, w hich enabled the identification of the developm ental processes, are show n.
First, a trichom e cell is selected from protoderm al cells. The try cpc (triptychon caprice) double m utant show s a defective trichom e
pattern. A cell that has adopted the trichom e cell fate sw itches from m itotic cycles to endoreduplication cycles. A situation in w hich
this sw itch is suppressed is exem plified by a trichom e in w hich a specific B -type cyclin, C YC B 1;2, is overexpressed under the
control of the trichom e-specific GL2prom oter (GL2:CYCB1;2). The glabra2(gl2) m utant phenotype indicates that cell-fate
determ ination requires specific gene functions. The angustifolia(an) m utant illustrates the requirem ent of genes to undergo proper
branching. The directionality of expansion grow th requires a group of several so-called DISTORTEDgenes, of w hich the wurm(wrm)
m utant is show n. The kaktus (kak) m utant has tw ice the D N A content of w ild-type plants and represents the class of m utants that
regulates the num ber of endoreduplication cycles. C ell-death control can also be studied using trichom es. C ertain m utants and
the overexpression of the cell-cycle kinase inhibitor/interactor of cyclin-dependent kinases (IC K) under the control of the trichom e-
specific GL2prom oter (GL2:ICK), w hich is show n here lead to unscheduled cell death. Finally, several m utants have a fragile and
glassy appearance and are therefore thought to be involved in the m aturation of the trichom e cell (not show n). The draw ings of
developm ental stages are adapted from REF. 6and the gl2 im age is reproduced from REF. 98. O ther im ages are reproduced w ith
perm ission as follow s: try cpc from REF. 19 (2002) M acm illan M agazines Ltd; GL2:CYCB1;2from REF. 35 (2002) Elsevier; anfrom
REF. 10 (1997) The C om pany of B iologists Ltd; wrmfrom REF. 76 (2003) SpringerVerlag G m bH ; kakfrom REF. 7 (1994)
Elsevier; GL2:ICK from REF. 91 (2003) The Am erican Society of Plant B iologists.
try cpc GL2:CYCB1;2 gl2 an wrm kak GL2:ICK
Trichom e selection
Sw itch from m itosis
to endoreduplication C ell-fate determ ination B ranching Expansion grow th
R egulation of the
num ber of
endoreduplication
cycles C ell-death control
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NATURE REVI EWS | MOLECULAR CELL BIOLOGY VOLUME 5 |JUNE 2004 | 4 7 3
R E V I E WS
function as positive regulators of trichome develop-
ment. Mutations in the GLABRA1(GL1) and TRANS-
PARENT TESTA GLABRA1(TTG1) genes each result in
the complete absence of trichomes
16,17
, whereas
GLABRA3(GL3) and ENHANCER OF GL3(EGL3)
function in a redundant manner gl3mutants exhibit
fewer trichomes compared with wild-type plants,
whereas gl3 egl3double mutants are devoid of tri-
chomes
18
. The trichome-suppressing genes are repre-
sented by three redundantly acting genes (see below).
Mutations in the TRIPTYCHON (TRY) gene result in
trichome clusters, mutations in the CAPRICE(CPC)
gene cause an increased number of trichomes
19,20
and a
single mutant of ENHANCER CAPRICE TRIPTY-
CHON1(ETC1), which is an enhancer of cpcand try
mutants, is indistinguishable from wild-type plants
21
.
Genetic analysis has established the functional rela-
tionships between the four positive factors. The findings
that co-overexpression of GL3 and EGL3, as well as GL3
together with GL1, can rescue the ttg1-mutant pheno-
type indicates that TTG1 functions upstream of these
genes and that the other three factors function together
at the same point in the pathway
18,22
. These data have
been confirmed at the molecular level.All trichome-pro-
moting genes, except for TTG1, encode putative tran-
scription factors. GL1encodes a MYB-RELATED TRANSCRIPTION
FACTOR
23
, GL3a BASIC HELIXLOOPHELIX (BHLH) FACTOR
22
, EGL3
is a close homologue of GL3(REF. 18), whereas TTG1
encodes a WD40 PROTEIN whose molecular function is
unknown
24
.Yeast two-hybrid data indicate that the four
positive factors form a transcriptional-activation com-
plex in which GL3 forms a homodimer that binds to
GL1 (REF. 18,22). The GL3 protein also binds to the TTG1
protein, but through a different domain. No direct
interaction was found between GL1 and TTG1 (REF. 22).
It is likely that GL1 and GL3 mediate the transcriptional
activation, as both proteins contain transcriptional-acti-
vation domains. This complex is expected to be active in
trichome precursor cells and be inactivated in all other
protodermis cells by one or more known negative regu-
lators of trichome initiation.
The negative regulators TRY, CPC and ETC1 all
belong to a small family of single-repeat MYB proteins
with no obvious transcriptional-activation domain
1921
.
Overexpressi on of any of these protei ns aboli shes
fluctuations, individual cells will gain higher levels of the
promoting factor, produce more of the suppressing fac-
tor and, in turn, inhibit the neighbouring cells more
strongly. Eventually, these cells would become commit-
ted to the trichome cell fate. For this mechanism to
work a number of criteria have to be met (for theoreti-
cal considerations, see BOX 1). First, the positive and the
negative regulators need to be involved in a feedback
loop with the activator activating the inhibitor and the
inhibitor inhibiting the activator. A second requirement
is that the inhibitor can move.
The genetic and molecular analysis of the trichome-
patterning genes is consistent with this model, although
little proof is available that could directly demonstrate
the patterning mechanism. Both the positive and the
negative regulator are represented by a group of several
factors (FIG. 2). Four of the trichome-patterning genes
MYB-RELATED TRANSCRIPTION
FACTOR
A transcription factor that
containsa DNA-binding
domain that showssequence
similarity to vMYB, thefirst-
described member of thisfamily.
BASIC HELIXLOOPHELIX
(BHLH) FACTOR
A protein that containstwo
-helicesseparated by a loop
(theHLH domain), which binds
DNA in a sequence-specific
manner.
WD40 PROTEIN
A 40-amino-acid-long protein
motif that containsa WD
dipeptideat itscarboxy
terminus. Thisdomain is
found in many functionally
diverseproteinsand mediates
proteinprotein interactions.
Figure 2 |Redundancy of trichome-patterning genes.The top row of phenotypes show s the
redundancy of the negative regulators of trichom e patterning. From left to right, w ild-type leaves,
the patterning defects of triptychon(try) single, try caprice (cpc) double and try cpc enhancer
caprice triptychon (etc1) triple m utants are show n. C om pared w ith
w ild-type plants, trym utants have only a few trichom e clusters that contain 23 trichom es. The
additional rem oval of C PC results in an increased cluster size (up to 40 trichom es) and m utations
in ETC 1 lead to the form ation of trichom e clusters w ith several hundred trichom es. It is
notew orthy that cpc single m utants (not show n) show a subtle increase of the trichom e density
and that etc1m utants show no phenotype. The low er row of phenotypes show s that G LAB R A3
(G L3) and EN H AN C ER O F G L3 (EG L3) are redundant. B oth single m utants show a slight
reduction in trichom e num ber com pared w ith w ild-type plants and the double m utant lacks
trichom es. The im ages are reproduced w ith perm ission as follow s: top row from REF. 21 (2004)
Elsevier; low er row from REF. 18 (2003) The C om pany of B iologists Ltd.
WT try try cpc try cpc etc1
WT gl3 egl3 gl3 egl3
B ox 1 | Theoretical model to explain two-dimensional patterning
A theoretical model that explains how a denovo
spacing pattern can be established, which is
similar to that observed for trichomes, was
formulated by Meinhardt and Gierer
95
. The
essence of this model is that an activator controls
the production of its own inhibitor, which can
function over a long distance. This, however, is
not sufficient to create the initial differences in a uniform field of cells. For this, it is necessary that the activator has self-
enhancing properties to form a so-called autocatalytic loop. This allows the rapid amplification of small differences that
are caused by the stochastic fluctuation of biomolecules to trigger further patterning. One non-intuitive prediction of this
model is that both the activator and the inhibitor show the highest expression at the same locations and not, as might be
assumed, at mutually exclusive locations. Three steps of a simulation of this model that results in a spacing pattern are
shown (see online supplementary information S1 (movie); courtesy of Hans Meinhardt).
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R E V I E WS
Trichome differentiation
The homeodomain leucine-zipper protein that is
encoded by the GL2 gene
29,30
is thought to translate the
cues that are provided by the patterning genes into cell-
specific differentiation of several epidermal cell types
including the seed coat, root hairs and trichomes
17,29,30
(BOX 2). Thi s i s documented by the fi ndi ng that co-
overexpression of GL1 and the maize Rgene product
(a homologue of GL3) results i n an i ncreased and
ectopic expression of GL2 (REF. 31). Although some evi-
dence indicates that GL2 might also have a role in tri-
chome patterning, most of the available data indicate
that GL2 triggers downstream differentiation events
32
.
The gl2-mutant-trichome phenotype is characterized
by undifferentiated trichomes that resemble the com-
bined phenotypes of various trichome-morphogenesis
genes, which indicates that GL2 activates trichome-spe-
cific differentiation genes
7,29
. Supporting evidence comes
from the root-hair system, where it was shown that GL2
regulates the gene that encodes phospholipase D1,
which, in turn, promotes root-hair differentiation
33
.
Cell-cycle control during trichome development
Trichome cell-cycle-regulation mutants affect either the
switch from mitotic cycles to endoreduplication cycles,
or the number of endoreduplication rounds, and
thereby the ploidy level (FIG. 3).
trichome formation. They seem to function in a highly
redundant manner: trymutants have small trichome
clusters consisting of two or three trichomes, try cpc
double mutants have large clusters of up to 40 trichomes
and in try cpc etc1triple mutants, fields of several hun-
dred trichomes are observed (FIG. 2)
7,19,21
. These negative
factors seem to interfere with the function of the tran-
scriptional-activation complex by a competition mech-
anism
25
. Three-hybrid analysis has shown that the
interaction between GL1 and GL3 is counteracted by
TRY, thereby disturbing the formation of the proposed
functional transcriptional-activation complex
26
.
How cellcell communication and a regulatory
feedback loop are achieved is, at present, unknown.
However, evidence is available for root-hair patterning
in A. thaliana, which requires a set of identical and
closely related genes (BOX 2). Here it was shown using
a green fluorescent protein (GFP)CPC fusion protein
that the negative regulator CPC moves between
cells, probably via PLASMODESMATA, which indicates that
travelling transcription factors can mediate cellcell
communication
27
. It was also shown that the positive
root-hair-patterning genes WEREWOLF(WER; a GL1
homologue) and GLABRA2(GL2) are involved in a
negative-feedback loop with CPC
28
. It is conceivable
that a similar mechanism operates during trichome
patterning (BOX 2).
CORTEX CELL
Thetissuebetween thevascular
bundleand theepidermis. In
A.thaliana, thisisa singlecell
layer.
PLASMODESMATA
Cellcell connectionsin plants
through which macromolecules,
including RNA and proteins, can
betransported in a regulated
manner.
B ox 2 | Comparison of trichome and root-hair patterning
Root hairs seem to be specified by position-dependent induction as root hairs are only formed over the cleft between two
underlying CORTEX CELLS(see figure, part a). Epidermal cells that are positioned on a cortex cell do not form root hairs.
Most of the genes (or their homologues) that are involved in root-hair patterning are also involved in trichome
development (see figure, part b). The proposed models for trichome and root-hair patterning are similar, but the
resulting phenotypes are different. Whereas those cells that eventually express GLABRA2 (GL2) in the shoot become
trichomes, GL2-expressing root epidermal cells adopt a non-root-hair cell fate. For both systems it is postulated that in
those cells that express the GL2 gene, a trimeric active complex is formed that consists of a MYB-related transcription
factor (GLABRA1 (GL1) in trichomes or WEREWOLF (WER) in roots), a basic helixloophelix protein (GLABRA3
(GL3) and the homologous ENHANCER OF GL3 (EGL3)) and a WD40 protein (TRANSPARENT TESTA GLABRA1
(TTG1)). This active complex is thought to activate GL2 and the expression of the negative regulators TRIPTYCHON
(TRY), CAPRICE (CPC) and CAPRICE TRIPTYCHON1 (ETC1). These travel into the neighbouring cells where they
compete with the MYB-related transcrption factor for binding to the complex, which causes the complex to become
inactivated. Identical or homologous proteins are shown in the same colour.
TTG 1
W ER W ER
TTG 1
G L1
G L1
TTG 1
TR Y,
C PC ,
ETC 1
TR Y,
C PC ,
ETC 1
TR Y,
C PC ,
ETC 1
TR Y,
C PC ,
ETC 1
TR Y,
C PC ,
ETC 1
G L2
G L2 G L2
Trichom e developm ent N o trichom e developm ent
N o root-hair developm ent R oot-hair developm ent
G L3,
EG L3
G L3,
EG L3
G L3,
EG L3
TTG 1
TR Y,
C PC ,
ETC 1
G L2
G L3,
EG L3
C ortex cell C ortex cell
b
a
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NATURE REVI EWS | MOLECULAR CELL BIOLOGY VOLUME 5 |JUNE 2004 | 4 7 5
R E V I E WS
mutants. By contrast, the initiation of mitotic cycles in
simmutants is independent of D-type cyclins
36
.
The number of trichome endoreduplication cycles is
affected in at least ten mutants that display either lower
or higher ploidy levels than normal. The picture that is
emerging from their genetic and molecular analysis
is that several different molecular pathways are involved
in the regulation of trichome endoreduplication cycles.
Regulation by patterning genes. Two of the patterning
genes that are descri bed above, GL3and TRY, also
functi on as posi tive and negative regulators of
endoreduplication cycles; trytrichomes have a DNA
content of 64C and different gl3alleles exist that have
either a reduced or an increased DNA content
7,26
. This
dual functi on rai ses the fasci nati ng possi bi li ty that
tri chome cell-fate choice is functionally linked with
cell-cycle regulation.
DNA-catenation-dependent endoreduplication. ROOT
HAIRLESS2 (RHL2) and HYPOCOTYL6 (HYP6) are
positive regulators of endoreduplication cycles in tri-
chomes and in other cell types
37
. Both are plant homo-
logues of the archaeal DNA TOPOISOMERASEVI complex.
These topoisomerases can DECATENATE DNA and pro-
mote ATP-dependent separation of entangled DNA
37
.
It is unclear whether the observed defect in the progres-
sion of trichome endoreduplication is due to a physical
block of further DNA replication or the activation of a
checkpoint that controls progression of the endoredu-
plication cycle.
Regulation by plant hormones. The plant hormone
gibberellin promotes endoreduplication cycles. In
spindly(spy) mutants, which exhibit a constitutive gib-
berellin response, trichomes have a DNA content of
64C (REF. 38). Conversely, in a mutant that is incapable
of gibberellin synthesis, ga1-3, no trichomes are
formed
39,40
.
Regulation by protein degradation. A class of four tri-
chome mutants, kaktus(kak), rastafari (rfi), poly-
chome(poc) and hirsute(hir), all show a very similar
phenotype: they all have a ploi dy level of 64C. The
cloni ng of the KAK gene revealed that i t encodes a
protei n wi th sequence si mi lari ty to a UBI QUI TI N E3
LIGASE
41,42
. It is therefore assumed that ubiquitin-regu-
lated protein degradation controls the progression of
endoreduplication.
Regulation by a cell-death pathway. Two lines of evi-
dence indicate that a pathway exists that controls both
the progressi on of endoredupli cati on cycles (and
mitotic cycles) as well as programmed cell death. First,
the CONSTITUTIVE PATHOGEN RESPONSE5
(CPR5) gene i s i nvolved i n both processes. Second,
overexpression of an inhibitor of the cell-cycle kinase
INHIBITOR/INTERACTOR OF CYCLIN-DEPEN-
DENT KINASES/KIP-RELATED PROTEINS
(I CK/KRP) leads to reduced ploi dy and early tri -
chome cell death (see below).
The SIAMESE(SIM) gene suppresses the swi tch
from mitotic divisions to endoreduplication cycles
34
.
In simmutants, trichomes are multicellular and con-
tain between 2 and 15 cells. If the first cycle is already
mitotic, two trichomes are formed instead of one; if
the switch to endoreduplication from mitotic divisions
is late, multicellular trichomes that are morphologi-
cally normal are formed. As SIMhas not been cloned
yet, little is known about the molecular mechanism
that is involved. However, some information is avail-
able from a different line of experiments in which the
role of known cell-cycle genes i n the control of
endoreduplication was tested. The expression of cell-
cycle genes that are normally not active in trichomes
was used to test the effects on trichome endoreduplica-
tion. To avoid organism-wide defects that could cause
sickness or even lethality, trichome-specific gene pro-
moters were used. B-TYPEAND D-TYPECYCLINScould trigger
the formation of multicellular trichomes, and B-type
cyclins are involved in the transition from G2 phase to
mitosis. The overexpression of a specific B-type cyclin,
CYCB1;2, is sufficient to switch from endoreduplica-
tion cycles to mitotic cycles, which indicates that B-type
cycli ns are i mportant for thi s regulatory step
35
.
Surprisingly, the overexpression of the D-type cyclin
CYCD3;1can also lead to the switch
36
. D-type cyclins
are thought to control the transition from the G1 to
the Sphase of the cell cycle in animals; but these results
indicate that, in plants, they have an additional func-
ti on i n regulati ng the entry i nto mi tosi s. In sim
mutants, CYCB1;2 is expressed in trichomes, which
indicates that SIM inhibits the expression of mitotic
cyclins. However, this is not its only function, as over-
expression of CYCB1;2 in a simmutant background
shows a much stronger phenotype than the si ngle
B-TYPEAND D-TYPECYCLINS
Cyclinsregulatecell-cycle
progression through
interactionswith cyclin-
dependent protein kinases.
B-typecyclinsregulateentry
into mitosis, whereasD-type
cyclinsareimportant in G1
phaseand, in plants, also for
entry into mitosis.
DNA TOPOISOMERASE
An enzymethat can cleaveand
religatetheDNA to allow a more
relaxed DNA configuration.
DECATENATE
During DNA replication, sister
duplex moleculesbecome
interlinked (catenated).
Decatenation istheseparation of
two entangled chromosomes.
UBIQUITIN E3 LIGASE
An enzymethat attaches
ubiquitin to a protein, thereby
marking it for degradation in the
proteasome.
Figure 3 | Regulation of the cell cycle in trichomes. The m itotic cell cycle proceeds
through four phases: M (m itosis), G 1 (gap 1), S (synthesis) and G 2. Endoreduplication cycles
cut the cell cycle short by skipping the G 2 and M phases. Processes that affect the num ber
of endoreduplication cycles (blue) w ere identified by m utant analysis. The factors that are
involved in the sw itch from m itosis to endoreduplication are indicated in red. O verexpression
studies have show n that the m itotic cyclin C YC B 1;2 is sufficient to trigger com plete cell
divisions. The SIAMESE(SIM) gene represses C YC B 1;2 as simm utants express C YC B 1;2 in
trichom es. Also C YC D 3;1 overexpression causes a sw itch from endoreduplication to m itosis.
In contrast to the situation w ith C YC B 1;2, how ever, the total num ber of
m itotic/endoreduplication cycles is drastically increased during C YC D 3;1 overexpression,
w hich indicates an additional role for this cyclin at G 1.
M
S
G 1
G 2
U biquitin-dependent
protein degradation
Patterning
D N A catenation
C ell death
C YC D 3;1
C YC B 1;2
SIAM ESE
H orm ones (gibberellin)
Endoreduplication
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R E V I E WS
branches
7,47
. The reduction of the ploidy level results in a
reduced branch number as found in cpr5, gl3 rhl2
and hyp6 (REFS7,37,48). It is likely that this observed con-
trol of trichome branching by the ploidy level is indirect,
and that the cell size or the time of actual cell growth
provides the frame for branch initiation.
Regulation by microtubules. Microtubules have an
important role in trichome branching, as shown in
experiments with microtubule antagonists. If the micro-
tubule cytoskeleton is defective during trichome growth,
the cell expands almost equally in all directions (this is
known as isotropic growth) and does not initiate
branches
49
. Several branching genes encode components
that are involved in the biogenesis of /-tubulin
dimers, the formation and stability of microtubules or
microtubule-based transport processes. Two weak
mutants of TUBULIN FOLDING COFACTOR (TFC)A
and TFCC which are involved in the correct folding
of tubulins and therefore the formation of assembly-
competent / tubulin dimers exhibit a bloated and
underbranched trichome phenotype
5052
. The analysis of
the microtubule cytoskeleton in these mutants sheds
some light on how the microtubules are reoriented dur-
ing branch initiation. As the microtubule density and
orientation is normal, it is likely that the failure of
branch formation is due to problems in denovosynthe-
sis rather than in the reorientation of pre-existing
microtubules. If branching requires the synthesis of
new microtubules, it is conceivable that it also requires
the fragmentation of pre-existing ones to allow a reori-
entation of growth. This view is supported by the
reduced-branching phenotype of mutants of the
KATANIN-P60gene
5355
. Katanins are known to cut pre-
existing microtubules into smaller fragments.
Microtubule reorientation during branch formation is
therefore assumed to be controlled by severing pre-
existing microtubules combined with denovosynthesis.
The spatial control of the orientation of micro-
tubules is regulated by at least two branching genes. The
FASS/TONNEAU2gene regulates mi crotubules i n
the context of cell divisions as can be inferred from
the observation that infass/tonneau2mutants cell-divi-
sion orientation is randomized
56,57
. It encodes a novel
protein, phosphatase-2A regulatory subunit, which
indicates that it regulates microtubules by protein
phosphorylation
58
. The second regulator of micro-
tubule organisation is SPIKE
59
. This protein shows
sequence similarity to CDM-family adaptor proteins
(Caenorhabditis elegans CED-5; Homo sapiens
DOCK180; Drosophila melanogaster MYOBLAST
CITY). These proteins function as guanine nucleotide-
exchange factors (GEFs)
60
and are thought to modulate
the cytoskeleton through small RHO-like GTPases
(known as ROPs in plants)
61
.
In addition, specific microtubule-based transport
processes seem to be important for branch formation.
The branching gene ZWICHEL (ZWI) encodes a
calmodulin-binding kinesin motor protein that binds
microtubules in a calmodulin-dependent manner
6267
.
The activity of ZWI is modulated by the KIC protein,
Trichome branching
The typical three-dimensional branching pattern of
trichomes is a unique model system for studying how
several axes of polari ty and cell morphogenesi s are
establi shed. Except for the simmutant, all mutants
descri bed so far affect the number of branches but
not thei r ori entati on wi th respect to each other.
Geneti c and molecular data i ndi cate that several
independent molecular pathways participate in tri-
chome branching
7,10,4346
(FIG. 4).
Regulation by endoreduplication levels. The number of
branches on a trichome depends on the ploidy level
of the cell. Tetraploid plants, in which the DNA con-
tent of all cells is doubled, have trichomes with super-
numerary branches
47
. Si mi larly, mutants that have
tri chomes with increased DNA levels such as kak,
poc, rfi, tryand spy have trichomes with up to eight
Figure 4 |Regulation of trichome branching. Trichom e branching is controlled by at least
four different m olecular processes. The m olecular analysis of the ANGUSTIFOLIA(AN) gene
indicates that transcriptional regulation and/or G olgi-related processes are im portant for
branching. The corresponding anm utant is underbranched. Several m utants that affect
m icrotubule function or organization have reduced branching; for exam ple, the tubulin folding
cofactor c (tfcc) m utant has tw o short branches. The branch num ber also correlates w ith the
ploidy levels. H igher ploidy levels lead to m ore branches, and trichom es w ith reduced ploidy
levels have few er branches. The triptychon(try) m utant show n here has tw ice the D N A content
of w ild-type cells and has five branches. A key regulator of branching is the STICHEL (STI)
gene, as the corresponding sti m utant trichom es do not initiate branches. H ow ever, the
biochem ical m echanism s through w hich STIC H EL regulates branching are, at present,
unknow n.The im ages are reproduced w ith perm ission as follow s: an, tryand sti from REF. 10
(1997) The C om pany of B iologists Ltd; tfcc from REF. 51 (2002) Elsevier; W T from REF. 76
(2003) SpringerVerlag G m bH .
G olgi/transcription M icrotubules Endoreduplication STI dependent
WT
an tfcc try sti
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NATURE REVI EWS | MOLECULAR CELL BIOLOGY VOLUME 5 |JUNE 2004 | 4 7 7
R E V I E WS
all the available data by assuming that branching is evo-
lutionarily derived from multicellular trichomes, in
which branching is the result of a certain division pat-
tern (BOX 3).
Directionality of trichome cell expansion
Like most plant cells, trichomes enlarge several-fold
during the later stages of differentiation and expand
in a polarized manner. This expansion occurs, unlike in
the growing tip of root hairs or pollen tubes, along the
whole cell surface
75,76
. The directionality of expansion
growth is affected in mutants of eight genes, which are
collectively known as the DISTORTEDgenes. All dis-
tortedmutants show a very si mi lar phenotype: tri -
chomes show turns and twi sts, some regi ons of the
cell become bulged and others are underdeveloped.
Following the movement of small beads that had been
placed on the trichome surface, it was shown that this
phenotype i s caused by the regi onally unbalanced
expansion of the cell
76
.
Findings from different experimental approaches
indicate that the directionality of trichome cell expansion
depends on the actin cytoskeleton. First, the application
of drugs that interfere with actin function perfectly phe-
nocopies the distortedmutant phenotype
75,77
. Second,
the actin cytoskeleton is organized aberrantly in dis-
tortedmutants (FIG. 5b)
7577
. Third, all DISTORTEDgenes
that have been cloned so far encode components of the
ARP2/3 COMPLEX
7881
, which promotes actin polymerization
by enhancing F-actin nucleation and side-binding activ-
ities that result in the initiation of fine actin filaments
from pre-existing F-actin
82,83
.
The analysis of the distortedmutants demonstrated
that actin has a role in expansion growth that goes
beyond its mere requirement for general growth. The
observation that in distortedmutants actin-based move-
ment of organelles, such as peroxisomes or the Golgi, is
not generally impaired indicates that F-actin is still
functional (see supplementary information S2(movie)
and S3(movie))
78,79
. Defects were found locally in those
parts of the cell that were not growing (FIG. 5d). Non-
growth regions contain heavily bundled actin, whereas
which binds to ZWI in a Ca
2+
-dependent manner
68
.
Thi s i ndi cates that ZWI-dependent transport
processes might ultimately be controlled by the intra-
cellular second messenger Ca
2+
.
Regulation by transcription or Golgi-related processes.
The ANGUSTIFOLIA(AN) gene regulates branching
by two possible pathways, by Golgi-related transport
processes or by transcri pti onal co-activati on. It
encodes a protein with sequence similarity to carboxy-
termi nal bi ndi ng protei n (CtBP) and brefeldi n-A-
ribosylated substrate (BARS)
69,70
. In D. melanogaster,
CtBP binds to the zinc-finger transcription factors and
functions as a transcriptional co-repressor
71
. In the rat,
BARS protei ns were i denti fi ed as protei ns that are
ADP-ribosylated after treatment with the fungal toxin
brefeldin A. Brefeldin-A treatments result in the trans-
formation of Golgi stacks into a tubular-reticular net-
work and it is therefore thought that BARSis involved
in Golgi functions
72,73
. Biochemical data are not avail-
able for the plant CtBP/BARS protein; however, the
findings that anmutants have microtubule defects and
that AN physically interacts with ZWI in a yeast two-
hybrid screen indicates that AN regulates microtubule
organization
69
.
Regulation by theSTICHEL gene. The STICHEL(STI)
gene regulates trichome branching in a dosage-depen-
dent manner; branch reduction is subtle in weak sti alle-
les, becomes more pronounced in stronger alleles and
trichomes are unbranched in null-alleles. Conversely,
overexpression of STI leads to extra branch formation
74
.
This genetic behaviour indicates a key regulatory role
for STI, although its molecular function is still elusive.
STI encodes a protein that contains a domain with
sequence similarity to eubacterial DNA-polymerase-III
subunits. However, it is unlikely that STI functions as a
DNA polymerase subunit, as no replication effects were
found to be associated with the branching phenotype.
An underlying scheme of how branch formation is
controlled is not evident from the current analysis of the
branching genes. One model, however, accommodates
PRE-PROPHASEBAND
A denseband of microtubulesat
thecell cortex that appears
beforethestart of cell division in
plants. Itsposition marksthe
futuredivision plane.
PHRAGMOPLAST
A fibrousstructurebetween the
daughter nuclei at telophasein
plant cells; also known asthecell
plate.
ARP2/3 COMPLEX
(Actin-related protein 2/3).
A multi-protein complex that
consistsof seven different
proteinsand initiatesnew actin
filamentson pre-existing ones.
B ox 3 | The evolutionary origin of trichome branching
In plant species other than Arabidopsisthaliana,
trichomes are frequently multicellular and branching is
initiated when cell division occurs in a plane that is
perpendicular to the main direction of growth
2
(see
figure). For example, the branches of the multicellular
and branched hairs in Verbascumare initiated by cell
divisions that are perpendicular to the main growth axis.
The finding that a single mutation in the SIAMESE (SIM)
gene is sufficient to produce multicellular trichomes in
A.thalianasupports such an evolutionary origin
34
. According to this model, all aspects concerning the establishment of
the orientation of the division plane and the corresponding cell polarity are still operating, even in the absence of the
actual cell division
43
. Several observations support this model, including the correlation of ploidy level and branch
number and the finding that the function of the kinesin motor protein ZWICHEL (ZWI) is linked to the cell cycle
96,97
.
ZWI is localized to the PRE-PROPHASEBAND and the PHRAGMOPLAST, and the injection of ZWI antibodies into multi-cellular
stamen hairs of Tradescantiavirginianaresults in a metaphase arrest and abnormal cell-plate formation
96,97
.
Figure adapted from REF. 2.
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R E V I E WS
RHOand RAC/CDC42signal-transduction pathways
that are known in animals and yeast are, in principle,
present in plants, although they are strongly modified. In
agreement with this, ROPs were shown to control the
local actin configuration in epidermal cells and down-
stream components, such as the HSPC300 (haematopo-
etic stem/progenitor-cell clone-300) complex, are known
to be involved in the control of actin organization
8486
.
Cell-death control in trichomes
The analysis of trichome development has revealed two
pathways that suppress cell death and also regulate
endoreduplication (see above). One pathway is repre-
sented by ICK/KRP, which shows homology to the ani-
mal cell-cycle inhibitor p27
Kip1
(REF. 87). In animals,
p27
Kip1
can induce apoptosis in the absence of growth
factors in some specific cell types
88
. When ubiquitously
expressed in the whole plant, ICK/KRP causes severe
growth reduction
87,89,90
, and when expressed under the
control of a tri chome-speci fi c promoter, tri chome
cells stop endoredupli cati on cycles after two cycles
and begin to die with symptoms that are characteristic
of programmed cell death, such as the degeneration of
CHROMOCENTRESand nucleoli
91
.
A second pathway is linked to the response of plants
to plant pathogens. A number of mutants mimic the
plant pathogen response. Many of these mutants show a
cell-death phenotype combined with growth defects
92
.
One of these mutants, cpr5, shows a trichome pheno-
type that is similar to that of ICK/KRP-overexpressing
lines; trichomes have a ploidy level of about 8C and
undergo unscheduled cell death
48
. It seems that in both
cases cell-cycle or endoreduplication-cycle progression
and the control of cell death are somehow linked; how-
ever, the mechanistic basis of this link remains to be
determined.
Control of maturation
Trichome maturation is affected in a group of diverse
mutants in which adult trichomes appear transparent
or underdeveloped. Three poorly characterized
mutants, chablis, chardonnayand retsina, have transpar-
ent trichomes and the underdeveloped trichome
mutant has no papilla on the trichome surface
93
. The
trichome birefringencemutant is defective in the pro-
duction of cellulose
94
.
Conclusions and perspectives
Almost all tri chome genes are i nvolved not only i n
trichome development, but also in the development
of other cell types and represent important compo-
nents of generally i mportant regulatory pathways.
The analysis of trichome initiation has uncovered an
evolutionarily conserved gene cassette of transcrip-
tion factors that are involved in patterning processes
and anthocyanin-synthesis control. Their evolution
and functional diversification will be very interesting
to study. Al so, the theoreti cal model that expl ai ns
pattern formation (BOX 1) is far from being proven; for
example, at present, there are no target promoters
known that could be used to test the genetic predictions.
regions in the distorted mutants that exhibit growth
comprise a fine network of actin known as fine actin. It
is conceivable that the creation of a local fine-actin net-
work promotes the transport of membrane and cell wall
material for the actual growth. It is speculated that the
actin cytoskeleton is also involved in the fusion of mem-
branes, as the fusion of small vacuoles, which normally
leads to the formation of the large central vacuole, does
not take place in distortedmutants
79
.
It is unknown how the ARP2/3-complex-dependent
formation of fine actin is spatially controlled in tri-
chomes. Some of the canonical pathways such as the
CHROMOCENTRE
A region in plant chromosomes
that comprisesheterochromatin
and coincideswith centromeres
during meiosis.
Figure 5 |Control of expansion polarity. Trichom e cell expansion is controlled by the actin
cytoskeleton. a| In w ild-type cells, actin is organized in long filam ents (as indicated by the arrow ).
b| M utants of the genes that are collectively know n as the DISTORTEDgenes exhibit fragm ented
actin (as indicated by the arrow ). All distorted-gene m utants, except for one, show the sam e
phenotype, w hich is a result of the fact that trichom e expansion is no longer coordinated. c | The
phenotype of one distorted-gene m utant, the wurmm utant, is show n as a scanning electron
m icroscope picture. d| Inside a cell of one of the distortedm utants, the crookedm utant, both
grow ing regions (left panel) and non-grow ing regions (right panel) show actin and G olgi vesicles.
The difference is that grow ing regions have fine actin, w hereas non-grow ing regions have bundled
actin. This indicates that fusion of vesicles w ith the m em brane is only prom oted in regions w ith
fine actin. O nline supplem entary inform ation S2 (m ovie) and S3 (m ovie) show actin-based
m ovem ent in peroxisom es, w hich, despite the actin-organization defects, is not generally affected
in the m utants (see online supplem entary inform ation S3 (m ovie)) com pared w ith the w ild-type
cells (see online supplem entary inform ation S2 (m ovie)). The im ages are reproduced w ith
perm ission as follow s: parts aand bfrom REF. 79 (2003) The Am erican Society of Plant
B iologists; part c from REF. 76 (2003) SpringerVerlag G m bH ; the im age in part dfrom REF. 78
(2003) The C om pany of B iologists Ltd.
a b c
d
2004 Nature PublishingGroup
NATURE REVI EWS | MOLECULAR CELL BIOLOGY VOLUME 5 |JUNE 2004 | 4 7 9
R E V I E WS
well as still unknown processes such as those controlled
by STI. Each group of genes has opened new research
areas in the plant sciences and it will be interesting to see
whether the common branching phenotype will tie these
processes together. With the discovery that cell-expan-
sion genes encode components of the ARP2/3 complex,
key components that regulate actin-based growth have
been identified and will allow the study of the up- and
downstream regulatory processes in plants. Further
analysis of trichomes as a single-cell model system offers
the chance to connect the above-mentioned, seemingly
unrelated, processesin the future.
Therefore, i t wi l l be chal l engi ng to show not onl y
that the i nhi bi tor protei ns can move, but also how
this is relevant for patterning.
Several pathways seem to have a role in how the
switch from mitosis to endoreduplication and the cycle
number are controlled. The isolation of further genes, in
combination with trichome-specific overexpression
approaches, should be a valuable addition to the cell-
cycle field. The analysis of branching genes has led to the
identification of proteinsthat are involved in processesas
different as intracellular transport, cell-size control, tran-
scriptional control and Golgi-dependent processes, as
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Acknow ledgem ents
I w ould like to thank H . M einhardt for providing the im ages and the
m ovie that are presented in BO X 1 and for stim ulating discussions. I
w ould also like to thank the m em bers of the laboratory for helpful
com m ents on the m anuscript. R esearch in the authors laboratory
is supported by the D eutsche Forschungsgem einschaft and the
Volksw agen Stiftung.
C om peting interests statem ent
The author declares that he has no com peting financialinterests.
Online links
DATABASES
The following terms in this article are linked online to:
TA I R : http://w w w .arabidopsis.org/
AN| CPC| CPR5| C YC B1;2 | C YC D 3;1 | EGL3| GL1| GL2| GL3|
H YP6 | KAK | R H L2 | SIM| STI | TTG1| WER| ZWI
SUPPLEMENTARY INFORMATION
S e e o n lin e a rtic le : S1 (m ovie) | S2 (m ovie) | S3 (m ovie)
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