In plants, trichomes have greatly improved our understanding of development at the single-cell level. A large number of mutants have been characterized that enabled the identification of subsequent developmental processes. The study of all the developmental stages of a single cell is a first step towards an understanding of how general cellular processes are integrated during development.
In plants, trichomes have greatly improved our understanding of development at the single-cell level. A large number of mutants have been characterized that enabled the identification of subsequent developmental processes. The study of all the developmental stages of a single cell is a first step towards an understanding of how general cellular processes are integrated during development.
In plants, trichomes have greatly improved our understanding of development at the single-cell level. A large number of mutants have been characterized that enabled the identification of subsequent developmental processes. The study of all the developmental stages of a single cell is a first step towards an understanding of how general cellular processes are integrated during development.
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 2004 Nature PublishingGroup 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). 2004 Nature PublishingGroup 4 7 4 | JUNE 2004 |VOLUME 5 www.nature.com/reviews/molcellbio 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 2004 Nature PublishingGroup 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 2004 Nature PublishingGroup 4 7 6 | JUNE 2004 |VOLUME 5 www.nature.com/reviews/molcellbio 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 2004 Nature PublishingGroup 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. 2004 Nature PublishingGroup 4 7 8 | JUNE 2004 |VOLUME 5 www.nature.com/reviews/molcellbio 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 1. Esau, K. Anatomy of Seed Plants (John W iley & Sons, N ew York, 1977). 2. U phof, J. C . T. Plant hairs (eds. Zim m erm ann, W . & O zenda, P. G .) (G ebr. B orntrger, B erlin, 1962). 3. Johnson, H . B . Plant pubescence: an ecological perspective. Bot. Rev. 41, 233258 (1975). 4. M auricio, R . & R ausher, M . D . Experim ental m anipulation of putative selective agents provides evidence for the role of natural enem ies in the evolution of plant defense. Evolution 51, 14351444 (1997). 5. M arks, M . D . M olecular genetic analysis of trichom e developm ent in Arabidopsis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 137163 (1997). 6. H lskam p, M ., Schnittger, A. & Folkers, U . Pattern form ation and cell differentiation: trichom es in Arabidopsis as a genetic m odel system . Int. Rev. Cytol. 186, 147178 (1999). 7. H lskam p, M ., M isera, S. & Jrgens, G . G enetic dissection of trichom e cell developm ent in Arabidopsis. Cell 76, 555566 (1994). Trichome development was dissected into discrete developmental steps using a systematic mutagenesis screen. 8. Larkin, J. C ., Young, N ., Prigge, M . & M arks, M . D . The control of trichom e spacing and num ber in Arabidopsis. Development122, 9971005 (1996). Elegant demonstration that trichome spacing does not involve cell lineage. 9. M elaragno, J. E., M ehrotra, B . & C olem an, A. W . R elationship betw een endopolyploidy and cell size in epiderm al tissue of Arabidopsis. Plant Cell 5, 16611668 (1993). 10. Folkers, U ., B erger, J. & H lskam p, M . C ell m orphogenesis of trichom es in Arabidopsis: differential control of prim ary and secondary branching by branch initiation regulators and cell grow th. Development124, 37793786 (1997). 11. Schnittger, A., Folkers, U ., Schw ab, B ., Jrgens, G . & H lskam p, M . G eneration of a spacing pattern: the role of TRIPTYCHONin trichom e patterning in Arabidopsis. Plant Cell 11, 11051116 (1999). 12. H lskam p, M . & Schnittger, A. Spatial regulation of trichom e form ation in Arabidopsis thaliana. Semin. Cell Dev. Biol. 9, 213220 (1998). 13. Scheres, B . Plant patterning: TRY to inhibit your neighbors. Curr. Biol. 12, R 804R 806 (2002). 14. Schiefelbein, J. C ell-fate specification in the epiderm is: a com m on patterning m echanism in the root and shoot. Curr. Opin. Plant Biol. 6, 7478 (2003). 15. Larkin, J. C ., B row n, M . L. & Schiefelbein, J. H ow do cells know w hat they w ant to be w hen they grow up? Lessons from epiderm al patterning in Arabidopsis. Annu. Rev. Plant Biol. 54, 403430 (2003). 16. Koornneef, M ., D ellaert, L. W . M . & van der Veen, J. H . EM S- and radiation-induced m utation frequencies at individual loci in Arabidopsis thaliana. Mutat. Res. 93, 109123 (1982). 17. Koornneef, M . The com plex syndrom e of ttgm utants. Arabidopsis Information Service18, 4551 (1981). 18. Zhang, F., G onzalez, A., Zhao, M ., Payne, C . T. & Lloyd, A. A netw ork of redundant bH LH proteins functions in all TTG 1-dependent pathw ays of Arabidopsis. Development 130, 48594869 (2003). Excellent analysis of how the many functions of TTG1 are mediated by a partially redundant network of bHLH and MYB transcription factors. 19. Schellm ann, S. et al. TR IPTYC H O N and C APR IC E m ediate lateral inhibition during trichom e and root hair patterning in Arabidopsis. EMBO J . 21, 50365046 (2002). 20. W ada, T., Tachibana, T., Shim ura, Y. & O kada, K. Epiderm al cell differentiation in Arabidopsis determ ined by a mybhom olog, CPC. Science277, 11131116 (1997). 21. Kirik, V., Sim on, M ., H lskam p, M . & Schiefelbein, J. The ENHANCER OF TRY AND CPC1(ETC1) gene acts redundantly w ith TRIPTYCHONand CAPRICEin trichom e and root hair cell patterning in Arabidopsis. Dev. Biol. 268, 506513 (2004). 22. Payne, C . T., Zhang, F. & Lloyd, A. M . G L3 encodes a bH LH protein that regulates trichom e developm ent in Arabidopsis through interaction w ith G L1 and TTG 1. Genetics 156, 13491362 (2000). 23. O ppenheim er, D . G ., H erm an, P. L., Sivakum aran, S., Esch, J. & M arks, M . D . A mybgene required for leaf trichom e differentiation in Arabidopsis is expressed in stipules. Cell 67, 483493 (1991). 24. W alker, A. R . et al. The TRANSPARENT TESTA GLABRA1 locus, w hich regulates trichom e differentiation and anthocyanin biosynthesis in Arabidopsis, encodes a W D 40 repeat protein. Plant Cell 11, 13371349 (1999). 25. Szym anski, D . B ., Lloyd, A. M . & M arks, M . D . Progress in the m olecular genetic analysis of trichom e intiation and m orphogenesis in Arabidopsis. Trends Plant Sci. 5, 214219 (2000). 26. Esch, J. J. et al. A contradictory GLABRA3allele helps define gene interactions controlling trichom e developm ent in Arabidopsis. Development130, 58855894 (2003). First insight into how the negative regulator TRY might counteract the GL1GL3TTG1 complex. Yeast three-hybrid analysis showed that TRY competes with GL1 for binding to GL3. 27. W ada, T. et al. R ole of a positive regulator of root hair developm ent, C APR IC E, in Arabidopsis root epiderm al cell differentiation. Development129, 54095419 (2002). 28. Lee, M . M . & Schiefelbein, J. C ell patterning in the Arabidopsis root epiderm is determ ined by lateral inhibition w ith feedback. Plant Cell 14, 611618 (2002). Elegant molecular-genetic study that is the first to show a regulatory feedback mechanism during root- hair patterning. 29. R erie, W . G ., Feldm ann, K. A. & M arks, M . D . The glabra 2 gene encodes a hom eo dom ain protein required for norm al trichom e developm ent in Arabidopsis. Genes Dev. 8, 13881399 (1994). 30. C ristina, M . D . et al. The Arabidopsis Athb-10 (G LAB R A2) is an H D -Zip protein required for regulation of root hair developm ent. Plant J . 10, 393402 (1996). 31. Szym anski, D . B ., Jilk, R . A., Pollock, S. M . & M arks, M . D . C ontrol of G L2 expression in Arabidopsis leaves and trichom es. Development125, 11611171 (1998). 32. O hashi, Y., R uberti, I., M orelli, G . & Aoyam a, T. Entopically additive expression of G LAB R A2 alters the frequency and spacing of trichom e initiation. Plant J . 21, 50365046 (2002). 33. O hashi, Y. et al. M odulation of phospholipid signaling by GLABRA2in root-hair pattern form ation. Science300, 14271430 (2003). Identification of the first gene that functions downstream of the patterning machinery. 34. W alker, J. D ., O ppenheim er, D . G ., C oncienne, J. & Larkin, J. C . SIAMESE, a gene controlling the endoreduplication cell cycle in Arabidopsis thaliana trichom es. Development127, 39313940 (2000). 35. Schnittger, A., Schobinger, U ., Stierhof, Y. D . & H lskam p, M . Ectopic B -type cyclin expression induces m itotic cycles in endoreduplicating Arabidopsis trichom es. Curr. Biol. 12, 415420 (2002). 36. Schnittger, A. et al. Ectopic D -type cyclin expression induced not only D N A replication but also cell division in Arabidopsis trichom es. Proc. Natl Acad. Sci. USA99, 64105415 (2002). Overexpression studies showed that plant D-type cyclins function, not only at the G1 transition, but also at the entry of mitosis. 37. Sugim oto-Shirasu, K., Stacey, N . J., C orsar, J., R oberts, K. & M cC ann, M . C . D N A topoisom erase VI is essential for endoreduplication in Arabidopsis. Curr. Biol. 12, 17821786 (2002). 38. Jacobsen, S. E., B inkow ski, K. A. & O lszew ski, N . E. SPIN D LY, a tetratricopeptide repeat protein involved in gibberellin signal transduction in Arabidopsis. Proc. Natl Acad. Sci. USA 93, 92929296 (1996). 39. C hien, J. C . & Sussex, I. M . D ifferential regulation of trichom e form ation on the adaxial and abaxial leaf surfaces by gibberellins and photoperiod in Arabidopsis thaliana (L.) H eynh. Plant Physiol. 111, 13211328 (1996). 40. Telfer, A., B ollm an, K. M . & Poethig, R . S. Phase change and the regulation of trichom e distribution in Arabidopsis thaliana. Development124, 645654 (1997). 41. D ow nes, B. P., Stupar, R. M ., G ingerich, D . J. & Vierstra, R. D . The H EC T ubiquitin-protein ligase (U PL) fam ily in Arabidopsis: U PL3 has a specific role in trichom e developm ent. Plant J . 35, 729742 (2003). 42. El R efy, A. et al. The Arabidopsis KAKTUS gene encodes a H EC T protein and controls the num ber of endoreduplication cycles. Mol. Genet. Genomics 270, 403414 (2004). 43. H lskam p, M . H ow plants split hairs. Curr. Biol. 10, R 308R 310 (2000). 44. Krishnakum ar, S. & O ppenheim er, D . G . Extragenic suppressors of the Arabidopsis zwi-3m utation identify new genes that function in trichom e branch form ation and pollen tube grow th. Development126, 30793088 (1999). 45. O ppenheim er, D . G enetics of plant cell shape. Curr. Opin. Plant Biol. 1, 520524 (1998). 46. Luo, D . & O ppenheim er, D . G . G enetic control of trichom e branch num ber in Arabidopsis: the roles of the FURCAloci. Development126, 55475557 (1999). Detailed genetic studies on the genetic interactions between the branching genes showed that they function largely in independent pathways. 47. Perazza, D . et al. Trichom e cell grow th in Arabidopsis thalianacan be depressed by m utations in at least five genes. Genetics 152, 461476 (1999). 48. Kirik, V. et al. C PR 5 is involved in cell proliferation and cell death control and encodes a novel transm em brane protein. Curr. Biol. 11, 18911895 (2001). 49. M athur, J. & C hua, N .-H . M icrotubule stabilization leads to grow th reorientation in Arabidopsis thalianatrichom es. Plant Cell 12, 465477 (2000). 50. Kirik, V. et al. The Arabidopsis TUBULIN-FOLDING COFACTORAgene is involved in the control of the /-tubulin m onom er balance. Plant Cell 14, 22652276 (2002). 51. Kirik, V. et al. Functional analysis of the tubulin-folding cofactor C in Arabidopsis thaliana. Curr. Biol. 12, 15191523 (2002). 52. Steinborn, K. et al. The Arabidopsis PILZgroup genes encode tubulin-folding cofactor orthologs required for cell division but not cell grow th. Genes Dev. 16, 959971 (2002). 53. B ichet, A., D esnos, T., Turner, S., G randjean, O . & H fte, H . B O TER O 1 is required for norm al orientation of cortical m icrotubules and anisotropic cell expansion in Arabidopsis. Plant J . 25, 137148 (2001). 2004 Nature PublishingGroup 4 8 0 | JUNE 2004 |VOLUME 5 www.nature.com/reviews/molcellbio R E V I E WS 54. W ebb, M ., Jouannic, S., Forem an, J., Linstead, P. & D olan, L. C ell specification in the Arabidopsis root epiderm is requires the activity of EC TO PIC R O O T H AIR 3 a katanin-p60 protein. Development129, 123131 (2002). 55. B urk, D . H ., Liu, B ., Zhong, R ., M orrison, W . H . & Ye, Z. H . A katanin-like protein regulates norm al cell w all biosynthesis and cell elongation. Plant Cell 13, 807827 (2001). 56. Torres-R uiz, R . A. & Jrgens, G . M utations in the FASS gene uncouple pattern form ation and m orphogenesis in Arabidopsis developm ent. Development120, 29672978 (1994). 57. Traas, J. et al. N orm al differentiation patterns in plants lacking m icrotubular preprophase bands. Nature375, 676677 (1995). 58. C am illeri, C . et al. The Arabidopsis TONNEAU2gene encodes a putative novel protein phosphatase 2A regulatory subunit essential for the control of the cortical cytoskeleton. Plant Cell 14, 833845 (2002). 59. Q iu, J. L., Jilk, R ., M arks, M . D . & Szym anski, D . B . The Arabidopsis SPIKE1gene is required for norm al cell shape control and tissue developm ent. Plant Cell 14, 101118 (2002). 60. B rugnera, E. et al. U nconventional R ac-G EF activity is m ediated through the D ock180ELM O com plex. Nature Cell Biol. 4, 574582 (2002). 61. D eeks, M . J. & H ussey, P. J. Arp2/3 and the shape of things to com e. Curr. Opin. Plant Biol. 6, 561567 (2003). Excellent review on the possible role and regulation of actin through the ARP2/3 complex. 62. R eddy, A. S., N arasim hulu, S. B ., Safadi, F. & G olovkin, M . A plant kinesin heavy chain-like protein is a calm odulin- binding protein. Plant J . 10, 921 (1996). 63. R eddy, A. S. N ., Safadi, F., N arasim hulu, S. B ., G olovkin, M . & H u, X. A novel plant calm odulin-binding protein w ith a kinesin heavy chain m otor dom ain. J . Biol. Chem. 271, 70527060 (1996). 64. R eddy, A. S. N ., N arasim hulu, S. B . & D ay, I. S. Structural organization of a gene encoding a novel calm odulin-binding kinesin-like protein from Arabidopsis. Gene204, 195200 (1997). 65. Song, H ., G olovkin, M ., R eddy, A. S. & Endow , S. A. In vitrom otility of AtKC B P, a calm odulin-binding kinesin protein of Arabidopsis. Proc. Natl Acad. Sci. USA 94, 322237 (1997). 66. D eavours, B . E., R eddy, A. S. & W alker, R . A. C a 2+ /calm odulin regulation of the Arabidopsis kinesin-like calm odulin-binding protein. Cell Motil. Cytoskeleton40, 408416 (1998). 67. O ppenheim er, D . G . et al. Essential role of a kinesin-like protein in Arabidopsis trichom e m orphogenesis. Proc. Natl Acad. Sci. USA 94, 62616266 (1997). 68. R eddy, V. S., D ay, I., Thom as, T. & R eddy, A. S. N . KIC , a novel C a 2+ binding protein w ith one EF-hand m otif, interacts w ith a m icrotubule m otor protein and regulates trichom e m orphogenesis. Plant Cell 16, 185200 (2004). 69. Folkers, U . et al. The cell m orphogenesis gene ANGUSTIFOLIAencodes a C tB P/B AR S-like protein and is involved in the control of the m icrotubule cytoskeleton. EMBO J . 21, 12801288 (2002). 70. Kim , G . T. et al. The ANGUSTIFOLIAgene of Arabidopsis, a plant C tB P gene, regulates leaf-cell expansion, the arrangem ent of cortical m icrotubules in leaf cells and expression of a gene involved in cell-w all form ation. EMBO J . 26, 12671279 (2002). 71. N ibu, Y., Zhang, H . & Levine, M . Interaction of a short-range repressors w ith DrosophilaC tB P in the em bryo. Science 280, 101104 (1998). 72. M atteis, M . D . et al. Stim ulation of endogenous AD P- ribosylation by brefeldin A. Proc. Natl Acad. Sci. USA 91, 11141118 (1994). 73. Lippincott-Schw artz, J., Yuan, L. C ., B onifacino, J. S. & Klausner, R . D . R apid redistribution of G olgi proteins into the ER in cells treated w ith brefeldin A: evidence for m em brane cycling from G olgi to ER . Cell 56, 801813 (1989). 74. Ilgenfritz, H . et al. The Arabidopsis STICHEL gene is a regulator of trichom e branch num ber and encodes a novel protein. Plant Physiol. 131, 643655 (2003). 75. Szym anski, D . B ., M arks, M . D . & W ick, S. M . O rganized F-actin is essential for norm al trichom e m orphogenesis in Arabidopsis. Plant Cell 11, 23312348 (1999). 76. Schw ab, B . et al. R egulation of cell expansion by the DISTORTEDgenes in Arabidopsis thaliana: actin controls the spatial organization of m icrotubules. Mol. Genet. Genomics 269, 350360 (2003). 77. M athur, J., Spielhofer, P., Kost, B . & C hua, N .-H . The actin cytoskeleton is required to elaborate and m aintain spatial patterning during trichom e cell m orphogenesis in Arabidopsis thaliana. Development126, 55595568 (1999). 78. M athur, J. et al. Arabidopsis CROOKED encodes for the sm allest subunit of the AR P2/3 com plex and controls cell shape by region specific fine F-actin form ation. Development130, 31373146 (2003). 79. M athur, J., M athur, N ., Kernebeck, B . & H lskam p, M . M utations in actin-related proteins 2 and 3 affect cell shape developm ent in Arabidopsis. Plant Cell 15, 16321645 (2003). 80. Le, J., El-Assal Sel, D ., B asu, D ., Saad, M . E. & Szym anski, D . B . R equirem ents for Arabidopsis ATAR P2 and ATAR P3 during epiderm al developm ent. Curr. Biol. 13, 13411347 (2003). 81. Li, S., B lanchoin, L., Yang, Z. & Lord, E. M . The putative Arabidopsis arp2/3 com plex controls leaf cell m orphogenesis. Plant Physiol. 132, 20342044 (2003). 82. M ullins, R . D ., H euser, J. A. & Pollard, T. D . The interaction of Arp2/3 com plex w ith actin: nucleation, high affinity pointed end capping, and form ation of branching netw orks of filam ents. Proc. Natl Acad. Sci. USA95, 61816186 (1998). 83. Svitkina, T. M . & B orisy, G . G . AR P2/3 com plex and actin depolym erizing factor/cofilin in dendritic organization and treadm illing of actin filam ent array in lam ellipodia. J . Cell Biol. 145, 10091026 (1999). 84. M athur, J. & H lskam p, M . Signal transduction: R ho-like proteins in plants. Curr. Biol. 12, R 526R 528 (2002). 85. Sm ith, L. G . C ytoskeletal control of plant cell shape: getting the fine points. Curr. Opin. Plant Biol. 6, 6373 (2003). 86. Yang, Z. Sm all G TPases: versatile signaling sw itches in plants. Plant Cell 14(Suppl.) 375388 (2002). 87. D e Veylder, L. et al. Functional analysis of cyclin-dependent kinase inhibitors of Arabidopsis. Plant Cell 13, 16531668 (2001). 88. H irom ura, K., Pippin, J. W ., Fero, M . L., R oberts, J. M . & Shankland, S. J. M odulation of apoptosis by the cyclin- dependent kinase inhibitor p27(Kip1). J . Clin. Invest. 103, 597604 (1999). 89. W ang, H . et al. IC K1, a cyclin-dependent protein kinase inhibitor from Arabidopsis thalianainteracts w ith both C dc2a and C ycD 3, and its expression is induced by abscisic acid. Plant J . 15, 501510 (1998). 90. Jasinski, S. et al. The C D K inhibitor N tKIS1a is involved in plant developm ent, endoreduplication and restores norm al developm ent of cyclin D 3;1-overexpressing plants. J . Cell Sci. 115, 973982 (2002). 91. Schnittger, A., W einl, C ., B ouyer, D ., Schobinger, U . & H lskam p, M . M isexpression of the cyclin-dependent kinase inhibitor IC K1/KR P1 in single-celled Arabidopsis trichom es reduces endoreduplication and cell size and induces cell death. Plant Cell 15, 303315 (2003). 92. G lazebrook, J. G enes controlling expression of defense responses in Arabidopsis 2001 status. Curr. Opin. Plant Biol. 4, 301308 (2001). 93. H aughn, G . W . & Som erville, C . R . G enetic control of m orphogenesis in Arabidopsis. Dev. Genet. 9, 7389 (1988). 94. Potikha, T. & D elm er, D . A m utant of Arabidopsis thaliana displaying altered patterns of cellulose deposition. Plant J . 7, 453460 (1995). 95. M einhardt, H . & G ierer, A. Applications of a theory of biological pattern form ation based on lateral inhibition. J . Cell Sci. 15, 321346 (1974). 96. Vos, J. W ., Safadi, F., R eddy, A. S. & H epler, P. K. The kinesin-like calm odulin binding protein is differentially involved in cell division. Plant Cell 12, 979990 (2000). 97. B ow ser, J. & R eddy, A. S. N . Localization of a kinesin-like calm odulin-binding protein in dividing cells of Arabidopsis and tobacco. Plant J . 12, 14291437 (1997). 98. Schw ab, B ., Folkers, U ., Ilgenfritz, H ., and H lskam p, M . Trichom e m orphogenesis in Arabidopsis. Philos. Trans. R. Soc. Lond. B355, 879883 (2000). 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) Access to this links box is available online. 2004 Nature PublishingGroup