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The patterning of epidermal hairs in Arabidopsis — updated
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The patterning of epidermal hairs in Arabidopsis — updated Markus Grebe Epidermal hairs of Arabidopsis thaliana emerge in regular spacing patterns providing excellent model systems for studies of biological pattern formation. A number of root-hair and leaftrichome patterning mutants and tools for cell-specific and tissue-specific manipulation of patterning protein activities have been combined in cycles of experimentation and mathematical modelling. These approaches have provided insight into molecular mechanisms of epidermal patterning. During the last two years, endoreplication has, unexpectedly, been found to control cell-fate maintenance during trichome patterning. New genetic interactions between a downstream, positive transcriptional regulator and lateral inhibitors of trichome or non-root-hair fate specification have been uncovered. A lateral inhibitor and a new positive regulator have been identified as major loci affecting trichome patterning in natural Arabidopsis populations. Finally, factors that modify root-hair patterning from the underlying cell layer have been discovered. Address Umea˚ Plant Science Centre, Department of Plant Physiology, Umea˚ University, SE-90187 Umea˚, Sweden Corresponding author: Grebe, Markus ([email protected])
Current Opinion in Plant Biology 2012, 15:31–37 This review comes from a themed issue on Growth and Development Edited by Xuemei Chen and Thomas Laux Available online 11th November 2011 1369-5266/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2011.10.010
Introduction Cells of multicellular organisms acquire distinct fates and undergo specific differentiation events. Cell-fate decisions and differentiation are coordinated in time and space in the process of pattern formation. Pattern formation can be easily studied in an organism by observing cells of the outer cell layer (epidermis) of an organism that acquire distinct shapes, such as the leaf hairs (trichomes), the root hairs and the stomata of plants. These cell types display regular spacing patterns in Arabidopsis thaliana [1– 5]. Here, I discuss recent findings on leaf-trichome and root-hair patterning that involve similar factors and mechanisms, whilst I refer readers to excellent recent reviews on stomatal patterning [6,7,8]. www.sciencedirect.com
One scenario to explain how a regular spacing pattern in a tissue layer can be established is that communication between initially equivalent cells promotes a specific fate in one cell and represses this fate in its neighbours. The establishment of leaf-trichome and root-hair cell fate largely follows this scenario [3,4], although positional cues from the underlying tissue layer are of additional importance in the root [9,10]. Trichome and root-hair patterning in Arabidopsis have been studied by molecular genetic methods for several decades and a large body of knowledge is available on gene regulatory networks involved in epidermal hair patterning [9,10,11,12,13]. At the core of these networks are positive and negative regulators of trichome and non-root-hair cell fate [9,10,11,12,13]. In a simplistic view, leaf pavement cell identity may be considered as a ground state of leaf epidermal cells in which trichome fate needs specification (Figure 1a,b). On the other hand, several factors that specify trichome fate in the shoot promote non-hair cell fate in the root. Accordingly, some mutants defective in positive regulators of trichome fate display glabrous leaves [14,15] but show excess formation of ectopic root hairs [1]. The upstream, positive regulators controlling trichome cell fate are GLABRA1 (GL1), an R2R3 MYB transcription factor [16], that acts in part redundantly with MYB23 [17], GLABRA3 (GL3), a basic helix–loop–helix transcription factor [18], its homologue ENHANCER OF GL3 (EGL3) [19,20], and TRANSPARENT TESTA GLABRA1 (TTG1), a WD40-repeat protein [15] (Figure 1c). These proteins also act as positive regulators of non-hair cell fate in the root epidermis [6], except for GL1, whose function is replaced by its homologues WEREWOLF (WER) and MYB23, here [21,22] (Figure 2). In the leaf, all positive regulators, except for MYB23, are initially expressed in all cells at the base of the leaf, where cell division activity is highest. Further up the leaf expression of the positive regulators becomes enhanced and confined to cells acquiring trichome fate [13]. The widely accepted model of lateral inhibition assumes that initial stochastic differences in the expression of positive regulators between cells subsequently become enhanced through the action of negative regulators of trichome cell fate that act on neighbouring cells [3,4]. Indeed, six such lateral inhibitors have been identified: TRIPTYCHON (TRY), CAPRICE (CPC) [23], ENHANCER OF TRY AND CPC (ETC) 1,2,3 [24–26] and TRICHOME-LESS (TCL) [27]. These are small, R3 single-repeat MYB transcriptional regulators that can bind to positive regulators and may inhibit the formation of activating regulatory complexes [24–29] (Figure 1c). In the root, as expected several of these proteins, in particular CPC, negatively regulate non-hair cell fate [9,28] (Figure 2c). Current Opinion in Plant Biology 2012, 15:31–37
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Once a threshold level of activator complex has been reached, this is thought to activate expression of downstream regulators. These include the homeodomain transcription factor GLABRA2 (GL2) a positive regulator of trichome cell fate (and non-root-hair cell fate) and trichome differentiation [14,30–32]. The activator complex, however, also induces expression of its R3 MYB inhibitors [30–33]. Depending on their binding affinities to the positive regulators, the R3 MYBs can, due to their smaller size, move laterally into neighbouring epidermal cells [12,29] (Figure 1c). Here, they are thought to antagonize assembly of positive regulators into activator complexes, thereby preventing cells from acquiring trichome or non-root-hair fate. Apart from lateral inhibition, additional mechanisms of interactions between fate activators and fate inhibitors contribute to trichome and roothair patterning [29,33–38], but I will not discuss them here in order to focus on more recent findings. I discuss the recent discovery that endoreplication cycles, which have so far been studied mainly for their importance in morphogenesis, contribute to maintenance of trichome cell fate; I present newly described interactions between R3 MYB inhibitors and GL2, as well as the identification of activators and inhibitors as major trait loci affecting trichome patterning in natural populations. Finally, I introduce novel factors regulating root-hair patterning from the underlying cortex cell layer.
A role for endoreplication in trichome patterning One of the early hallmarks of trichome cell differentiation is the entry of the cell into an endoreplication cycle which during subsequent enlargement of the cell and the formation of trichome branches is followed by two to three additional rounds of endoreplication [39] (Figure 1a). Strikingly, the positive fate regulator GL3 and the lateral inhibitor TRY also have opposite effects on endoreplication; gl3 mutants lack one cycle of endoreplication whilst try mutants undergo one additional cycle [39]. Mutants with reduced endoreplication typically have less branched trichomes, whilst mutants with additional endoreplication cycles often display more trichome branches [39]. Therefore, endoreplication has been mostly recognized for its role in trichome morphogenesis. A recent study, however, shows that a reduction of endoreplication cycles by interference with cell cycle control is accompanied by a decrease in the numbers of leaf trichomes [40], suggesting that endoreplication also has a role in trichome patterning. The siamese (sim) mutant, for example, is defective in an inhibitor of cyclin-dependent kinases (CDKs) required for endoreplication and undergoes multicellularization instead of endoreplication of trichomes but at the same time it forms fewer trichomes per leaf [40,41]. SIM is very likely a direct target for GL3 control of endoreplication during the maintenance of trichome cell fate; GL1 and GL3 bind the SIM promoter in chromatin immunoprecipitation (CHIP) experiments [33] and SIM Current Opinion in Plant Biology 2012, 15:31–37
expression is downregulated in trichomes of gl3 but slightly upregulated in trichomes of try mutants [42]. Moreover, similar patterning defects of gl2 gl3 and gl2 sim double mutants further indicate that GL3 and SIM act in the same pathway during trichome patterning [40]. Similarly to SIM loss-of-function, overexpression of the CDK activator CYCLIND3;1 (CYCD3;1) from the late GL2 promoter promotes multicellularization of trichomes and reduces the number of trichomes formed per leaf [40,43], whilst expressing CYCD3;1 from early promoters completely abolishes trichome initiation [40]. These findings could mean that overexpression of the cell division activator CYCD3;1 or loss of the inhibitor, SIM, interfere with trichome fate establishment by promoting cell division of an incipient trichome cell [40]. This could prevent accumulation of the fate activator complex to a critical threshold level, thus hindering cells from acquiring trichome fate. Alternatively, endoreplication could promote or maintain trichome fate [40]. Several lines of evidence suggest that this is the case. For instance, when the CDK inhibitor ICK1/KRP1 is overexpressed late during trichome fate specification, endoreplication is reduced [40,44]; already initiated trichomes abort and GL2 promoter activity ceases in these aborting cells [40], indicating that they are loosing trichome fate. On the other hand, the trichome-cell specific overexpression of an activator of endoreplication, CCS52A1 [45,46], in a gl2 gl3 double mutant results in formation of trichome-like structures on the leaves of this glabrous mutant [40]. Consistent with these results, loss of ccs52A1 function enhances the multicellular trichome phenotype of the sim mutant [45], although patterning defects for ccs52A1 loss-of-function mutants have not yet been reported. Certainly, this work provides a prime example for how endoreplication can contribute to cell-fate maintenance during pattern formation (Figure 1c). The potential direct link between patterning and endoreplication can now be further explored. In this respect it is interesting to note that GL1 and GL3 bind the RETINOBLASTOMA RELATED (RBR) gene promoter in CHIP experiments [33], reduction of RBR function results in increased endoreplication and in the formation of trichomes with multiple branches [47]. It will thus be exciting to see whether RBR has a role in trichome patterning. It also remains puzzling and worthwhile investigating how GL1/GL3, on the one hand, regulate SIM expression needed for endoreplication, but on the other hand potentially also regulate RBR which inhibits endoreplication in trichomes. It has also been hypothesized that a link between the cell cycle machinery, RBR, and GL1 may exist at the level of the regulatory sequences of the GL1 gene [48]. These contain potential bindings sites for E2F, a transcription factor that regulates expression of genes involved in DNA replication and is directly inhibited by RBR [47]. These examples may illustrate the potential for exploring putative feedback between the regulation of endoreplication and trichome patterning (Figure 1c). www.sciencedirect.com
The patterning of epidermal hairs in Arabidopsis — updated Grebe 33
Figure 1
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A model for trichome patterning including a role for endoreplication in cell-fate maintenance. (a) Cartoon of a young Arabidopsis leaf with the leaf base and the youngest cells to the bottom, more differentiated trichomes with three branches to the top. Cells acquiring trichome fate start to inhibit neighbours from becoming trichomes (red inhibitory lines). Nuclear DNA content of pavement cells is 2c, but trichome cells undergo up to four endoreplication cycles (4c, 8c, 16c, 32c) as indicated (blue). (b) Scanning electron micrograph of a two-week-old wild-type leaf displaying regularly spaced trichomes with three to four branches, leaf base to the bottom. (c) Simplified trichome patterning model. GL1/MYB23, GL3/EGL3 and TTG1 form fate activator complex(es) triggering trichome fate. MYC1 is a likely additional component of this complex. The activator complex directly activates transcription (black arrows) of its inhibitors TRY, CPC, ETC2 and other R3 MYBs that undergo protein movement (red arrows) to neighbouring cells. Here, they replace GL1 in complexes keeping them inactive so that the cell remains a non-trichome cell. Note, TTG1 can move into trichome cells and is trapped by GL3 [35]. The activator complex is thought to be autoactivating (pattern formation loop) and also directly activates transcription of GL2, a downstream regulator of trichome fate and differentiation, as well as transcription of the mitosis inhibitor SIM required for endoreplication. Endoreplication is important for maintenance of trichome fate in a feedback loop between SIM-dependent endoreplication and GL2 action. This likely involves CCS52A1 and potentially RBR (not shown).
New interactions between presumptive downstream regulators and lateral inhibitors A well-established function of GL3 is its action as a selector in the fate activator complex in concert with R2R3 MYBs and TTG1. GL3 directly binds to the promoters of several single-repeat R3 MYB inhibitors, www.sciencedirect.com
namely CPC, TRY, ETC1 and ETC3 (also called CPL3), and to the promoter of GL2 in CHIP experiments [33] and it activates the expression of several of these genes [12,13,31–33]. The relationship between the singlerepeat R3 MYB inhibitors and GL2, however, remains unclear. The recent finding that gl2 try cpc triple mutants Current Opinion in Plant Biology 2012, 15:31–37
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Figure 2
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A simplified model for root-hair patterning including non-cell autonomous signals from the cortex. (a) Root tip of an Arabidopsis seedling with files of non-hair cells (N) colour-coded in blue and files of hair cells (H) in red. (b) Cartoon displaying radial organization of cell types in the root tip in one half of a cross section corresponding to the inset in a (dashed line). Cell types displayed are epidermal (epi) H cells (red) and N cells (blue), ground tissue divided in cortex (co) and endodermis (en) surrounding vascular tissue cells (vas). Inset of dashed line marks the area displayed in c. (c) Model for roothair patterning. Epidermal N cells (blue), H cells (red) and cortical cells (white). WER/MYB23, GL3/EGL3 and TTG1 form non-hair fate activator complex(es) triggering non-hair cell fate. The activator complex expressed in non-hair cells directly activates transcription (black arrows) of its inhibitors CPC, TRY and ETC1 that undergo protein movement (red arrows) to neighbouring cells. Here, they replace WER/MYB23 in complexes keeping them inactive so that the cell can acquire root-hair fate. Note, GL3 proteins can move into non-hair cells (green arrows). In cells in the N position, the activator complex directly activates transcription of GL2 that represses hair cell fate in N cells. The SCM leucine-rich repeat receptor kinase-like protein indirectly represses WER transcription in cells in the H position and acts in one pathway with JKD. JKD acts non-cell autonomously from the ground tissue on epidermal patterning. SCZ acts cell autonomously to repress hair formation in cortical cells and non-cell autonomously on cell-fate separation in epidermal cells.
form a high number of root hairs in ectopic positions, displaying a hairy phenotype similar to gl2 single mutants, suggests that GL2 is epistatic to these single-repeat R3 MYB genes in root-hair patterning [49]. Hence, TRY and CPC likely act in one pathway with GL2 in the root epidermis; the situation, nevertheless, is different in the shoot. Here, try cpc and etc1 are able to suppress the glabrous leaf phenotype of gl2 with increasing efficiency in higher-order mutant combinations, suggesting that R3 MYBs do not solely act through GL2 [49]. This means that, in addition to GL2 expression, the R3 MYBs inhibitors apparently modulate other events, still to be discovered, during trichome fate specification.
New insight from trichome patterning in natural populations Trichome patterning has mostly been analysed by forward and reverse genetic approaches. Yet, variation of trichome density in natural populations can also be Current Opinion in Plant Biology 2012, 15:31–37
exploited to identify loci affecting trichome patterning, although a potential selective advantage of differences in trichome density is sometimes difficult to determine in natural populations [50]. Nevertheless, recent analyses of natural variation in trichome density led to some surprising discoveries. The single-repeat R3 MYB inhibitor gene ETC2 was found to be responsible for a substantial part of the variation in trichome number observed across 75 Arabidopsis accessions [51]. This was unexpected because etc2 knockout mutants, previously isolated from the widely used laboratory accessions Col or WS, displayed very modest defects in trichome patterning. Yet, a single-nucleotide polymorphism in ECT2 was found responsible for the low-trichome-number phenotype in the Gr-1 accession compared to the high trichome number in the Can-0 accession. The authors speculate that this mutation in a conserved but previously uncharacterized domain of the protein may enhance stability of the ECT2 repressor resulting in reduced trichome numbers. www.sciencedirect.com
The patterning of epidermal hairs in Arabidopsis — updated Grebe 35
Intriguingly, the Col and WS accessions do not contain the ETC2 allele with the strong repressor function [51]. Thus, isolation of knockout mutants in these standard laboratory backgrounds could not reveal the strong effect of ETC2 on trichome patterning observed by analysis of natural variation. A second study on natural variation identified AtMYC1, encoding a presumptive transcriptional regulator, as a locus required for trichome patterning. A single-nucleotide polymorphism in AtMYC1, responsible for the low-trichome-density phenotype observed in four different Arabidopsis accessions, was found to create a proline-to-alanine substitution in AtMYC1 [52]. Intriguingly, this mutation abolished interaction of AtMYC1 with the TTG1 and GL1 proteins observed in a yeast two-hybrid system [52]. Hence, this study on natural allelic variation identified a function for AtMYC1 in trichome patterning and a new protein domain required for interaction with trichome-fate activators. Interestingly, AtMYC1 was also found as a potential direct target gene for GL1 and GL3 in CHIP experiments [33]. Together, these findings open the door for analysis of AtMYC1 function within the gene regulatory network of trichome patterning.
Signals from below in root-hair patterning The patterning of hair and non-hair cells in the root epidermis shares mechanisms and players with trichome patterning. The fate regulators common to both systems, however, have opposite functions in root and shoot. The GL1 homologue WER, for example, interacts with GL3 and TTG1 in non-hair cells to specify non-hair fate in the root, whilst single-repeat R3 MYB inhibitors like CPC act as repressors of non-hair fate (or activators of hair fate) [9,10,12]. Moreover, root-hair formation is organized in files of hair (H) cells and non-hair (N) cells along the longitudinal axis of the root. This involves positional information from the underlying cortical cell layer. Trichoblasts form in the cleft between two underlying cortical cells and differentiate into H cells, whilst epidermal cells overlying a single cortical cell become atrichoblasts and normally acquire N-cell fate [9,10] (Figure 2). The molecular factors that may act from the cortex to specify epidermal hair fate, however, remained unknown. The SCRAMBLED (SCM) leucine-rich receptor kinase-like protein is a candidate for transmitting a presumptive cue for trichoblast fate from the cortical layer [9,53]. scm mutants display a randomized patchy pattern of hair formation and of epidermal WER expression that normally becomes confined to atrichoblasts and is only ubiquitously expressed in early meristematic epidermal cells [9,53,54]. Interestingly, double mutant analysis places SCM in the same pathway as JACKDAW (JKD) [54]. This is further supported by the findings that expression of WER, CPC and GL2 is randomized in epidermal cells of jkd and scm single mutants [54]. This came as a surprise because the JKD zinc finger protein is not expressed in epidermal but in cortical, endodermal www.sciencedirect.com
and quiescent centre cells. In the ground tissue, the endodermis and the cortex, JKD is known to regulate asymmetric cell division and the range of action of proteins specifying cell fate [55]. Strikingly, however, expressing JKD in cortical cells of the jkd mutant is sufficient to correct the epidermal patterning defects, strongly suggesting that JKD acts on epidermal patterning in a non-cell autonomous manner [54]. Yet, a second factor contributing to epidermal cell-fate determination from underlying tissue layers has recently been discovered. The schizoriza (scz) mutant was originally identified based on a high number of root hairs formed in the N position and, intriguingly, the formation of root hairs from cells of the underlying cortical layer [56]. Now, SCZ has been found to encode HEAT SHOCK TRANSCRIPTION FACTOR B4 (HSFB4) that in the postembryonic root is expressed in the quiescent centre, the ground tissue initials, the endodermis and cortex, but not in the epidermis [57,58]. Both SCZ loss-of-function and overexpression affect asymmetric division required for cell-fate separation and induce expression of hair and non-hair fate or differentiation markers in cortex cells, demonstrating that SCZ is a regulator of cell-fate separation [57,58]. SCZ appears to act cell autonomously on cell-fate segregation in the ground tissue but non-cell autonomously on patterning of root epidermal cells [57]. The identification of non-cell autonomous functions for the JKD and SCZ genes is certainly exciting. These molecular players now provide stepping stones for analyses of how positional information from the cell layer below interacts with the gene regulatory network of pattern formation in and between root epidermal cells.
Competing interest statement The author declares that he has no competing interests.
Acknowledgements M.G. thanks Stefano Pietra and the reviewers for helpful comments on the manuscript as well as Arp Schnittger for discussion on the role of endoreplication in cell-fate maintenance. M.G. further acknowledges Per Ho¨rstedt for providing Figure 1b and Yoshihisa Ikeda for providing the root image for Figure 2a. The author apologizes to those colleagues whose relevant work has not been cited due to space limitations. M.G.’s research on the Arabidopsis root epidermis is supported by the Swedish Research Council (Vetenskapsra˚det) and by an ERC Starting Researcher Grant 2010 from the European Research Council.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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25. Kirik V, Simon M, Wester K, Schiefelbein J, Hu¨lskamp M: ENHANCER of TRY and CPC 2 (ETC2) reveals redundancy in the region-specific control of trichome development of Arabidopsis. Plant Mol Biol 2004, 55:389-398. 26. Tominaga R, Iwata M, Sano R, Inoue K, Okada K: Arabidopsis C, APRICE-LIKE MYB 3 (CPL3) controls endoreduplication and flowering development in addition to trichome and root hair formation. Development 2008, 135:1335-1345. 27. Wang S, Kwak SH, Zeng Q, Ellis BE, Chen XY, Schiefelbein J, Chen JG: TRICHOMELESS1 regulates trichome patterning by suppressing GLABRA1 in Arabidopsis. Development 2007, 134:3873-3882. 28. Wang S, Hubbard L, Chang Y, Guo J, Schiefelbein J, Chen JG: Comprehensive analysis of single-repeat R3 MYB proteins in epidermal cell patterning and their transcriptional regulation in Arabidopsis. BMC Plant Biol 2008, 8:81. 29. Wester K, Digiuni S, Geier F, Timmer J, Fleck C, Hu¨lskamp M: Functional diversity of R3 single-repeat genes in trichome development. Development 2009, 136:1487-1496. 30. Szymanski DB, Jilk RA, Pollock SM, Marks MD: Control of GL2 expression in Arabidopsis leaves and trichomes. Development 1998, 125:1161-1171. 31. Wang S, Chen JG: Arabidopsis transient expression analysis reveals that activation of GLABRA2 may require concurrent binding of GLABRA1 and GLABRA3 to the promoter of GLABRA2. Plant Cell Physiol 2008, 49:1792-1804. 32. Zhao M, Morohashi K, Hatlestad G, Grotewold E, Lloyd A: The TTG1–bHLH–MYB complex controls trichome cell fate and patterning through direct targeting of regulatory loci. Development 2008, 135:1991-1999. 33. Morohashi K, Grotewold E: A systems approach reveals regulatory circuitry for Arabidopsis trichome initiation by the GL3 and GL1 selectors. PLoS Genet 2009, 5:e1000396. 34. Savage NS, Walker T, Wieckowski Y, Schiefelbein J, Dolan L, Monk NA: A mutual support mechanism through intercellular movement of CAPRICE and GLABRA3 can pattern the Arabidopsis root epidermis. PLoS Biol 2008, 6:e235. 35. Bouyer D, Geier F, Kragler F, Schnittger A, Pesch M, Wester K, Balkunde R, Timmer J, Fleck C, Hu¨lskamp M: Two-dimensional patterning by a trapping/depletion mechanism: the role of TTG1 and GL3 in Arabidopsis trichome formation. PLoS Biol 2008, 6:e141. 36. Digiuni S, Schellmann S, Geier F, Greese B, Pesch M, Wester K, Dartan B, Mach V, Srinivas BP, Timmer J et al.: A competitive complex formation mechanism underlies trichome patterning on Arabidopsis leaves. Mol Syst Biol 2008, 4:217. 37. Benı´tez M, Alvarez-Buylla ER: Dynamic-module redundancy confers robustness to the gene regulatory network involved in hair patterning of Arabidopsis epidermis. Biosystems 2010, 102:11-15. 38. Benı´tez M, Espinosa-Soto C, Padilla-Longoria P, AlvarezBuylla ER: Interlinked nonlinear subnetworks underlie the formation of robust cellular patterns in Arabidopsis epidermis: a dynamic spatial model. BMC Syst Biol 2008, 2:98. www.sciencedirect.com
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39. Hu¨lskamp M, Misera S, Ju¨rgens G: Genetic dissection of trichome cell development in Arabidopsis. Cell 1994, 76:555-566. 40. Bramsiepe J, Wester K, Weinl C, Roodbarkelari F, Kasili R, Larkin JC, Hu¨lskamp M, Schnittger A: Endoreplication controls cell fate maintenance. PLoS Genet 2010, 6:e1000996. This is the first study directed to test for and reveal a connection between endoreplication and cell-fate maintenance based on developmental stage-specific modulation of the endoreplication machinery employing overexpression and downregulation of cell cycle regulators. 41. Churchman ML, Brown ML, Kato N, Kirik V, Hu¨lskamp M, Inze´ D, De Veylder L, Walker JD, Zheng Z, Oppenheimer DG et al.: SIAMESE, a plant-specific cell cycle regulator, controls endoreplication onset in Arabidopsis thaliana. Plant Cell 2006, 18:3145-3157. 42. Jakoby MJ, Falkenhan D, Mader MT, Brininstool G, Wischnitzki E, Platz N, Hudson A, Hu¨lskamp M, Larkin J, Schnittger A: Transcriptional profiling of mature Arabidopsis trichomes reveals that NOECK encodes the MIXTA-like transcriptional regulator MYB106. Plant Physiol 2008, 148:1583-1602. 43. Schnittger A, Scho¨binger U, Bouyer D, Weinl C, Stierhof YD, Hu¨lskamp M: Ectopic D-type cyclin expression induces not only DNA replication but also cell division in Arabidopsis trichomes. Proc Natl Acad Sci U S A 2002, 99:6410-6415. 44. Schnittger A, Weinl C, Bouyer D, Scho¨binger U, Hu¨lskamp M: Misexpression of the cyclin-dependent kinase inhibitor ICK1/ KRP1 in single-celled Arabidopsis trichomes reduces endoreduplication and cell size and induces cell death. Plant Cell 2003, 15:303-315. 45. Kasili R, Walker JD, Simmons LA, Zhou J, De Veylder L, Larkin JC: SIAMESE cooperates with the CDH1-like protein CCS52A1 to establish endoreplication in Arabidopsis thaliana trichomes. Genetics 2010, 185:257-268. See annotation to Ref. [46]. 46. Larson-Rabin Z, Li Z, Masson PH, Day CD: FZR2/CCS52A1 expression is a determinant of endoreduplication and cell expansion in Arabidopsis. Plant Physiol 2009, 149:874-884. This paper along with Ref. [45] do not address pattern formation but neatly establish the role of CCS52A1 in endoreplication by loss-of-function and gain-of-function studies [45,46] and especially also in its interaction with SIM through double-mutant analysis [46] which also aids the understanding of CCS52A1 and SIM function in trichome fate maintenance [40]. 47. Desvoyes B, Ramirez-Parra E, Xie Q, Chua NH, Gutierrez C: Cell type-specific role of the retinoblastoma/E2F pathway during Arabidopsis leaf development. Plant Physiol 2006, 140:67-80. 48. Wenger JP, Marks MD: E2F and retinoblastoma related proteins may regulate GL1 expression in developing Arabidopsis trichomes. Plant Signal Behav 2008, 3:420-422. 49. Wang S, Barron C, Schiefelbein J, Chen JG: Distinct relationships between GLABRA2 and single-repeat R3 MYB transcription factors in the regulation of trichome and root hair patterning in Arabidopsis. New Phytol 2010, 185:387-400. This study employs multiple mutant analyses which reveals unexpected interactions between GL2 and the single-repeat R3 MYB transcriptional regulators suggesting that they act through factors in addition to GL2 which now need to be discovered.
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50. Kawagoe T, Shimizu KK, Kakutani T, Kudoh H: Coexistence of trichome variation in a natural plant population: a combined study using ecological and candidate gene approaches. PLoS One 2011, 6:e22184. 51. Hilscher J, Schlo¨tterer C, Hauser MT: A single amino acid replacement in ETC2 shapes trichome patterning in natural Arabidopsis populations. Curr Biol 2009, 9:1747-1751. This work based on QTL mapping and subsequent comparison of a large number of ETC2 alleles from natural populations reveals new mutations in a previously uncharacterized domain of ETC2 that enhance or reduce its repressor activity. It demonstrates the power of analyses of natural allelic variation both for our understanding of molecular function and of the contribution of loci to patterning in natural populations. 52. Symonds VV, Hatlestad G, Lloyd AM: Natural allelic variation defines a role for ATMYC1: trichome cell fate determination. PLoS Genet 2011, 7:e1002069. This study based on QTL mapping and subsequent comparison of a large number of alleles from natural populations identifies AtMYC1 as a novel regulator of trichome patterning in Arabidopsis. The causative SNP discovered at the AtMYC1 locus from several low-trichome-density accessions also abolishes the protein interactions of AtMYC1 with GL1 and TTG1 discovered in the yeast two-hybrid system. The work reveals the first known function of AtMYC1. 53. Kwak SH, Schiefelbein J: A feedback mechanism controlling SCRAMBLED receptor accumulation and cell-type pattern in Arabidopsis. Curr Biol 2008, 18:1949-1954. 54. Hassan H, Scheres B, Blilou I: JACKDAW controls epidermal patterning in the Arabidopsis root meristem through a noncell-autonomous mechanism. Development 2010, 137:1523-1529. This report identifies the zinc finger protein JKD as one of the first two factors contributing positional information to root epidermal hair patterning from the underlying cortical cell layer, and it suggests that JKD acts in one pathway with the leucine-rich receptor kinase-like protein SCM. 55. Welch D, Hassan H, Blilou I, Immink R, Heidstra R, Scheres B: Arabidopsis JACKDAW and MAGPIE zinc finger proteins delimit asymmetric cell division and stabilize tissue boundaries by restricting SHORT-ROOT action. Genes Dev 2007, 21:2196-2204. 56. Mylona P, Linstead P, Martienssen R, Dolan L: SCHIZORIZA controls an asymmetric cell division and restricts epidermal identity in the Arabidopsis root. Development 2002, 129:4327-4334. 57. ten Hove CA, Willemsen V, de Vries WJ, van Dijken A, Scheres B, Heidstra R: SCHIZORIZA encodes a nuclear factor regulating asymmetry of stem cell divisions in the Arabidopsis root. Curr Biol 2010, 20:452-457. See annotation to Ref. [58]. 58. Pernas M, Ryan E, Dolan L: SCHIZORIZA controls tissue system complexity in plants. Curr Biol 2010, 20:818-823. This paper along with Ref. [57] independently identifies a role for the SCZ protein in cell-fate separation and Ref. [57] further demonstrates that SCZ acts non-cell autonomously on root epidermal patterning. These are also the first studies to identify a role for a HEAT SHOCK TRANSCRIPTION FACTOR B4 in cell-fate separation and patterning. They open new horizons for our understanding of specific functions of such proteins during development.
Current Opinion in Plant Biology 2012, 15:31–37
The patterning of epidermal hairs in Arabidopsis — updated Markus Grebe Epidermal hairs of Arabidopsis thaliana emerge in regular spacing patterns providing excellent model systems for studies of biological pattern formation. A number of root-hair and leaftrichome patterning mutants and tools for cell-specific and tissue-specific manipulation of patterning protein activities have been combined in cycles of experimentation and mathematical modelling. These approaches have provided insight into molecular mechanisms of epidermal patterning. During the last two years, endoreplication has, unexpectedly, been found to control cell-fate maintenance during trichome patterning. New genetic interactions between a downstream, positive transcriptional regulator and lateral inhibitors of trichome or non-root-hair fate specification have been uncovered. A lateral inhibitor and a new positive regulator have been identified as major loci affecting trichome patterning in natural Arabidopsis populations. Finally, factors that modify root-hair patterning from the underlying cell layer have been discovered. Address Umea˚ Plant Science Centre, Department of Plant Physiology, Umea˚ University, SE-90187 Umea˚, Sweden Corresponding author: Grebe, Markus ([email protected])
Current Opinion in Plant Biology 2012, 15:31–37 This review comes from a themed issue on Growth and Development Edited by Xuemei Chen and Thomas Laux Available online 11th November 2011 1369-5266/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2011.10.010
Introduction Cells of multicellular organisms acquire distinct fates and undergo specific differentiation events. Cell-fate decisions and differentiation are coordinated in time and space in the process of pattern formation. Pattern formation can be easily studied in an organism by observing cells of the outer cell layer (epidermis) of an organism that acquire distinct shapes, such as the leaf hairs (trichomes), the root hairs and the stomata of plants. These cell types display regular spacing patterns in Arabidopsis thaliana [1– 5]. Here, I discuss recent findings on leaf-trichome and root-hair patterning that involve similar factors and mechanisms, whilst I refer readers to excellent recent reviews on stomatal patterning [6,7,8]. www.sciencedirect.com
One scenario to explain how a regular spacing pattern in a tissue layer can be established is that communication between initially equivalent cells promotes a specific fate in one cell and represses this fate in its neighbours. The establishment of leaf-trichome and root-hair cell fate largely follows this scenario [3,4], although positional cues from the underlying tissue layer are of additional importance in the root [9,10]. Trichome and root-hair patterning in Arabidopsis have been studied by molecular genetic methods for several decades and a large body of knowledge is available on gene regulatory networks involved in epidermal hair patterning [9,10,11,12,13]. At the core of these networks are positive and negative regulators of trichome and non-root-hair cell fate [9,10,11,12,13]. In a simplistic view, leaf pavement cell identity may be considered as a ground state of leaf epidermal cells in which trichome fate needs specification (Figure 1a,b). On the other hand, several factors that specify trichome fate in the shoot promote non-hair cell fate in the root. Accordingly, some mutants defective in positive regulators of trichome fate display glabrous leaves [14,15] but show excess formation of ectopic root hairs [1]. The upstream, positive regulators controlling trichome cell fate are GLABRA1 (GL1), an R2R3 MYB transcription factor [16], that acts in part redundantly with MYB23 [17], GLABRA3 (GL3), a basic helix–loop–helix transcription factor [18], its homologue ENHANCER OF GL3 (EGL3) [19,20], and TRANSPARENT TESTA GLABRA1 (TTG1), a WD40-repeat protein [15] (Figure 1c). These proteins also act as positive regulators of non-hair cell fate in the root epidermis [6], except for GL1, whose function is replaced by its homologues WEREWOLF (WER) and MYB23, here [21,22] (Figure 2). In the leaf, all positive regulators, except for MYB23, are initially expressed in all cells at the base of the leaf, where cell division activity is highest. Further up the leaf expression of the positive regulators becomes enhanced and confined to cells acquiring trichome fate [13]. The widely accepted model of lateral inhibition assumes that initial stochastic differences in the expression of positive regulators between cells subsequently become enhanced through the action of negative regulators of trichome cell fate that act on neighbouring cells [3,4]. Indeed, six such lateral inhibitors have been identified: TRIPTYCHON (TRY), CAPRICE (CPC) [23], ENHANCER OF TRY AND CPC (ETC) 1,2,3 [24–26] and TRICHOME-LESS (TCL) [27]. These are small, R3 single-repeat MYB transcriptional regulators that can bind to positive regulators and may inhibit the formation of activating regulatory complexes [24–29] (Figure 1c). In the root, as expected several of these proteins, in particular CPC, negatively regulate non-hair cell fate [9,28] (Figure 2c). Current Opinion in Plant Biology 2012, 15:31–37
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Once a threshold level of activator complex has been reached, this is thought to activate expression of downstream regulators. These include the homeodomain transcription factor GLABRA2 (GL2) a positive regulator of trichome cell fate (and non-root-hair cell fate) and trichome differentiation [14,30–32]. The activator complex, however, also induces expression of its R3 MYB inhibitors [30–33]. Depending on their binding affinities to the positive regulators, the R3 MYBs can, due to their smaller size, move laterally into neighbouring epidermal cells [12,29] (Figure 1c). Here, they are thought to antagonize assembly of positive regulators into activator complexes, thereby preventing cells from acquiring trichome or non-root-hair fate. Apart from lateral inhibition, additional mechanisms of interactions between fate activators and fate inhibitors contribute to trichome and roothair patterning [29,33–38], but I will not discuss them here in order to focus on more recent findings. I discuss the recent discovery that endoreplication cycles, which have so far been studied mainly for their importance in morphogenesis, contribute to maintenance of trichome cell fate; I present newly described interactions between R3 MYB inhibitors and GL2, as well as the identification of activators and inhibitors as major trait loci affecting trichome patterning in natural populations. Finally, I introduce novel factors regulating root-hair patterning from the underlying cortex cell layer.
A role for endoreplication in trichome patterning One of the early hallmarks of trichome cell differentiation is the entry of the cell into an endoreplication cycle which during subsequent enlargement of the cell and the formation of trichome branches is followed by two to three additional rounds of endoreplication [39] (Figure 1a). Strikingly, the positive fate regulator GL3 and the lateral inhibitor TRY also have opposite effects on endoreplication; gl3 mutants lack one cycle of endoreplication whilst try mutants undergo one additional cycle [39]. Mutants with reduced endoreplication typically have less branched trichomes, whilst mutants with additional endoreplication cycles often display more trichome branches [39]. Therefore, endoreplication has been mostly recognized for its role in trichome morphogenesis. A recent study, however, shows that a reduction of endoreplication cycles by interference with cell cycle control is accompanied by a decrease in the numbers of leaf trichomes [40], suggesting that endoreplication also has a role in trichome patterning. The siamese (sim) mutant, for example, is defective in an inhibitor of cyclin-dependent kinases (CDKs) required for endoreplication and undergoes multicellularization instead of endoreplication of trichomes but at the same time it forms fewer trichomes per leaf [40,41]. SIM is very likely a direct target for GL3 control of endoreplication during the maintenance of trichome cell fate; GL1 and GL3 bind the SIM promoter in chromatin immunoprecipitation (CHIP) experiments [33] and SIM Current Opinion in Plant Biology 2012, 15:31–37
expression is downregulated in trichomes of gl3 but slightly upregulated in trichomes of try mutants [42]. Moreover, similar patterning defects of gl2 gl3 and gl2 sim double mutants further indicate that GL3 and SIM act in the same pathway during trichome patterning [40]. Similarly to SIM loss-of-function, overexpression of the CDK activator CYCLIND3;1 (CYCD3;1) from the late GL2 promoter promotes multicellularization of trichomes and reduces the number of trichomes formed per leaf [40,43], whilst expressing CYCD3;1 from early promoters completely abolishes trichome initiation [40]. These findings could mean that overexpression of the cell division activator CYCD3;1 or loss of the inhibitor, SIM, interfere with trichome fate establishment by promoting cell division of an incipient trichome cell [40]. This could prevent accumulation of the fate activator complex to a critical threshold level, thus hindering cells from acquiring trichome fate. Alternatively, endoreplication could promote or maintain trichome fate [40]. Several lines of evidence suggest that this is the case. For instance, when the CDK inhibitor ICK1/KRP1 is overexpressed late during trichome fate specification, endoreplication is reduced [40,44]; already initiated trichomes abort and GL2 promoter activity ceases in these aborting cells [40], indicating that they are loosing trichome fate. On the other hand, the trichome-cell specific overexpression of an activator of endoreplication, CCS52A1 [45,46], in a gl2 gl3 double mutant results in formation of trichome-like structures on the leaves of this glabrous mutant [40]. Consistent with these results, loss of ccs52A1 function enhances the multicellular trichome phenotype of the sim mutant [45], although patterning defects for ccs52A1 loss-of-function mutants have not yet been reported. Certainly, this work provides a prime example for how endoreplication can contribute to cell-fate maintenance during pattern formation (Figure 1c). The potential direct link between patterning and endoreplication can now be further explored. In this respect it is interesting to note that GL1 and GL3 bind the RETINOBLASTOMA RELATED (RBR) gene promoter in CHIP experiments [33], reduction of RBR function results in increased endoreplication and in the formation of trichomes with multiple branches [47]. It will thus be exciting to see whether RBR has a role in trichome patterning. It also remains puzzling and worthwhile investigating how GL1/GL3, on the one hand, regulate SIM expression needed for endoreplication, but on the other hand potentially also regulate RBR which inhibits endoreplication in trichomes. It has also been hypothesized that a link between the cell cycle machinery, RBR, and GL1 may exist at the level of the regulatory sequences of the GL1 gene [48]. These contain potential bindings sites for E2F, a transcription factor that regulates expression of genes involved in DNA replication and is directly inhibited by RBR [47]. These examples may illustrate the potential for exploring putative feedback between the regulation of endoreplication and trichome patterning (Figure 1c). www.sciencedirect.com
The patterning of epidermal hairs in Arabidopsis — updated Grebe 33
Figure 1
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A model for trichome patterning including a role for endoreplication in cell-fate maintenance. (a) Cartoon of a young Arabidopsis leaf with the leaf base and the youngest cells to the bottom, more differentiated trichomes with three branches to the top. Cells acquiring trichome fate start to inhibit neighbours from becoming trichomes (red inhibitory lines). Nuclear DNA content of pavement cells is 2c, but trichome cells undergo up to four endoreplication cycles (4c, 8c, 16c, 32c) as indicated (blue). (b) Scanning electron micrograph of a two-week-old wild-type leaf displaying regularly spaced trichomes with three to four branches, leaf base to the bottom. (c) Simplified trichome patterning model. GL1/MYB23, GL3/EGL3 and TTG1 form fate activator complex(es) triggering trichome fate. MYC1 is a likely additional component of this complex. The activator complex directly activates transcription (black arrows) of its inhibitors TRY, CPC, ETC2 and other R3 MYBs that undergo protein movement (red arrows) to neighbouring cells. Here, they replace GL1 in complexes keeping them inactive so that the cell remains a non-trichome cell. Note, TTG1 can move into trichome cells and is trapped by GL3 [35]. The activator complex is thought to be autoactivating (pattern formation loop) and also directly activates transcription of GL2, a downstream regulator of trichome fate and differentiation, as well as transcription of the mitosis inhibitor SIM required for endoreplication. Endoreplication is important for maintenance of trichome fate in a feedback loop between SIM-dependent endoreplication and GL2 action. This likely involves CCS52A1 and potentially RBR (not shown).
New interactions between presumptive downstream regulators and lateral inhibitors A well-established function of GL3 is its action as a selector in the fate activator complex in concert with R2R3 MYBs and TTG1. GL3 directly binds to the promoters of several single-repeat R3 MYB inhibitors, www.sciencedirect.com
namely CPC, TRY, ETC1 and ETC3 (also called CPL3), and to the promoter of GL2 in CHIP experiments [33] and it activates the expression of several of these genes [12,13,31–33]. The relationship between the singlerepeat R3 MYB inhibitors and GL2, however, remains unclear. The recent finding that gl2 try cpc triple mutants Current Opinion in Plant Biology 2012, 15:31–37
34 Growth and Development
Figure 2
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Current Opinion in Plant Biology
A simplified model for root-hair patterning including non-cell autonomous signals from the cortex. (a) Root tip of an Arabidopsis seedling with files of non-hair cells (N) colour-coded in blue and files of hair cells (H) in red. (b) Cartoon displaying radial organization of cell types in the root tip in one half of a cross section corresponding to the inset in a (dashed line). Cell types displayed are epidermal (epi) H cells (red) and N cells (blue), ground tissue divided in cortex (co) and endodermis (en) surrounding vascular tissue cells (vas). Inset of dashed line marks the area displayed in c. (c) Model for roothair patterning. Epidermal N cells (blue), H cells (red) and cortical cells (white). WER/MYB23, GL3/EGL3 and TTG1 form non-hair fate activator complex(es) triggering non-hair cell fate. The activator complex expressed in non-hair cells directly activates transcription (black arrows) of its inhibitors CPC, TRY and ETC1 that undergo protein movement (red arrows) to neighbouring cells. Here, they replace WER/MYB23 in complexes keeping them inactive so that the cell can acquire root-hair fate. Note, GL3 proteins can move into non-hair cells (green arrows). In cells in the N position, the activator complex directly activates transcription of GL2 that represses hair cell fate in N cells. The SCM leucine-rich repeat receptor kinase-like protein indirectly represses WER transcription in cells in the H position and acts in one pathway with JKD. JKD acts non-cell autonomously from the ground tissue on epidermal patterning. SCZ acts cell autonomously to repress hair formation in cortical cells and non-cell autonomously on cell-fate separation in epidermal cells.
form a high number of root hairs in ectopic positions, displaying a hairy phenotype similar to gl2 single mutants, suggests that GL2 is epistatic to these single-repeat R3 MYB genes in root-hair patterning [49]. Hence, TRY and CPC likely act in one pathway with GL2 in the root epidermis; the situation, nevertheless, is different in the shoot. Here, try cpc and etc1 are able to suppress the glabrous leaf phenotype of gl2 with increasing efficiency in higher-order mutant combinations, suggesting that R3 MYBs do not solely act through GL2 [49]. This means that, in addition to GL2 expression, the R3 MYBs inhibitors apparently modulate other events, still to be discovered, during trichome fate specification.
New insight from trichome patterning in natural populations Trichome patterning has mostly been analysed by forward and reverse genetic approaches. Yet, variation of trichome density in natural populations can also be Current Opinion in Plant Biology 2012, 15:31–37
exploited to identify loci affecting trichome patterning, although a potential selective advantage of differences in trichome density is sometimes difficult to determine in natural populations [50]. Nevertheless, recent analyses of natural variation in trichome density led to some surprising discoveries. The single-repeat R3 MYB inhibitor gene ETC2 was found to be responsible for a substantial part of the variation in trichome number observed across 75 Arabidopsis accessions [51]. This was unexpected because etc2 knockout mutants, previously isolated from the widely used laboratory accessions Col or WS, displayed very modest defects in trichome patterning. Yet, a single-nucleotide polymorphism in ECT2 was found responsible for the low-trichome-number phenotype in the Gr-1 accession compared to the high trichome number in the Can-0 accession. The authors speculate that this mutation in a conserved but previously uncharacterized domain of the protein may enhance stability of the ECT2 repressor resulting in reduced trichome numbers. www.sciencedirect.com
The patterning of epidermal hairs in Arabidopsis — updated Grebe 35
Intriguingly, the Col and WS accessions do not contain the ETC2 allele with the strong repressor function [51]. Thus, isolation of knockout mutants in these standard laboratory backgrounds could not reveal the strong effect of ETC2 on trichome patterning observed by analysis of natural variation. A second study on natural variation identified AtMYC1, encoding a presumptive transcriptional regulator, as a locus required for trichome patterning. A single-nucleotide polymorphism in AtMYC1, responsible for the low-trichome-density phenotype observed in four different Arabidopsis accessions, was found to create a proline-to-alanine substitution in AtMYC1 [52]. Intriguingly, this mutation abolished interaction of AtMYC1 with the TTG1 and GL1 proteins observed in a yeast two-hybrid system [52]. Hence, this study on natural allelic variation identified a function for AtMYC1 in trichome patterning and a new protein domain required for interaction with trichome-fate activators. Interestingly, AtMYC1 was also found as a potential direct target gene for GL1 and GL3 in CHIP experiments [33]. Together, these findings open the door for analysis of AtMYC1 function within the gene regulatory network of trichome patterning.
Signals from below in root-hair patterning The patterning of hair and non-hair cells in the root epidermis shares mechanisms and players with trichome patterning. The fate regulators common to both systems, however, have opposite functions in root and shoot. The GL1 homologue WER, for example, interacts with GL3 and TTG1 in non-hair cells to specify non-hair fate in the root, whilst single-repeat R3 MYB inhibitors like CPC act as repressors of non-hair fate (or activators of hair fate) [9,10,12]. Moreover, root-hair formation is organized in files of hair (H) cells and non-hair (N) cells along the longitudinal axis of the root. This involves positional information from the underlying cortical cell layer. Trichoblasts form in the cleft between two underlying cortical cells and differentiate into H cells, whilst epidermal cells overlying a single cortical cell become atrichoblasts and normally acquire N-cell fate [9,10] (Figure 2). The molecular factors that may act from the cortex to specify epidermal hair fate, however, remained unknown. The SCRAMBLED (SCM) leucine-rich receptor kinase-like protein is a candidate for transmitting a presumptive cue for trichoblast fate from the cortical layer [9,53]. scm mutants display a randomized patchy pattern of hair formation and of epidermal WER expression that normally becomes confined to atrichoblasts and is only ubiquitously expressed in early meristematic epidermal cells [9,53,54]. Interestingly, double mutant analysis places SCM in the same pathway as JACKDAW (JKD) [54]. This is further supported by the findings that expression of WER, CPC and GL2 is randomized in epidermal cells of jkd and scm single mutants [54]. This came as a surprise because the JKD zinc finger protein is not expressed in epidermal but in cortical, endodermal www.sciencedirect.com
and quiescent centre cells. In the ground tissue, the endodermis and the cortex, JKD is known to regulate asymmetric cell division and the range of action of proteins specifying cell fate [55]. Strikingly, however, expressing JKD in cortical cells of the jkd mutant is sufficient to correct the epidermal patterning defects, strongly suggesting that JKD acts on epidermal patterning in a non-cell autonomous manner [54]. Yet, a second factor contributing to epidermal cell-fate determination from underlying tissue layers has recently been discovered. The schizoriza (scz) mutant was originally identified based on a high number of root hairs formed in the N position and, intriguingly, the formation of root hairs from cells of the underlying cortical layer [56]. Now, SCZ has been found to encode HEAT SHOCK TRANSCRIPTION FACTOR B4 (HSFB4) that in the postembryonic root is expressed in the quiescent centre, the ground tissue initials, the endodermis and cortex, but not in the epidermis [57,58]. Both SCZ loss-of-function and overexpression affect asymmetric division required for cell-fate separation and induce expression of hair and non-hair fate or differentiation markers in cortex cells, demonstrating that SCZ is a regulator of cell-fate separation [57,58]. SCZ appears to act cell autonomously on cell-fate segregation in the ground tissue but non-cell autonomously on patterning of root epidermal cells [57]. The identification of non-cell autonomous functions for the JKD and SCZ genes is certainly exciting. These molecular players now provide stepping stones for analyses of how positional information from the cell layer below interacts with the gene regulatory network of pattern formation in and between root epidermal cells.
Competing interest statement The author declares that he has no competing interests.
Acknowledgements M.G. thanks Stefano Pietra and the reviewers for helpful comments on the manuscript as well as Arp Schnittger for discussion on the role of endoreplication in cell-fate maintenance. M.G. further acknowledges Per Ho¨rstedt for providing Figure 1b and Yoshihisa Ikeda for providing the root image for Figure 2a. The author apologizes to those colleagues whose relevant work has not been cited due to space limitations. M.G.’s research on the Arabidopsis root epidermis is supported by the Swedish Research Council (Vetenskapsra˚det) and by an ERC Starting Researcher Grant 2010 from the European Research Council.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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36 Growth and Development
3.
Schnittger A, Folkers U, Schwab B, Ju¨rgens G, Hu¨lskamp M: Generation of a spacing pattern: the role of TRIPTYCHON in trichome patterning in Arabidopsis. Plant Cell 1999, 11:1105-1116.
4.
Lee MM, Schiefelbein J: Cell pattern in the Arabidopsis root epidermis determined by lateral inhibition with feedback. Plant Cell 2002, 14:611-618.
22. Kang YH, Kirik V, Hu¨lskamp M, Nam KH, Hagely K, Lee MM, Schiefelbein J: The MYB23 gene provides a positive feedback loop for cell fate specification in the Arabidopsis root epidermis. Plant Cell 2009, 21:1080-1094.
5.
Geisler M, Nadeau J, Sack FD: Oriented asymmetric divisions that generate the stomatal spacing pattern in Arabidopsis are disrupted by the too many mouths mutation. Plant Cell 2000, 12:2075-2086.
23. Schellmann S, Schnittger A, Kirik V, Wada T, Okada K, Beermann A, Thumfahrt J, Ju¨rgens G, Hu¨lskamp M: TRIPTYCHON and CAPRICE mediate lateral inhibition during trichome and root hair patterning in Arabidopsis. EMBO J 2002, 21:5036-5046.
6. Dong J, Bergmann DC: Stomatal patterning and development. Curr Top Dev Biol 2010, 91:267-297. See annotation to Ref. [8].
24. Kirik V, Simon M, Hu¨lskamp M, Schiefelbein J: The ENHANCER OF TRY AND CPC1 gene acts redundantly with TRIPTYCHON and CAPRICE in trichome and root hair cell patterning in Arabidopsis. Dev Biol 2004, 268:506-513.
7.
Peterson KM, Rychel AL, Torii KU: Out of the mouths of plants: the molecular basis of the evolution and diversity of stomatal development. Plant Cell 2010, 22:296-306. See annotation to Ref. [8].
8.
Shimada T, Sugano SS, Hara-Nishimura I: Positive and negative peptide signals control stomatal density. Cell Mol Life Sci 2011, 68:2081-2088. This paper along with Refs. [6,7] provides three most recent reviews covering different aspects of stomatal patterning which are not discussed here due to space constraints.
9.
Schiefelbein J, Kwak SH, Wieckowski Y, Barron C, Bruex A: The gene regulatory network for root epidermal cell-type pattern formation in Arabidopsis. J Exp Bot 2009, 60:1515-1521.
10. Tominaga-Wada R, Ishida T, Wada T: New insights into the mechanism of development of Arabidopsis root hairs and trichomes. Int Rev Cell Mol Biol 2011, 286:67-106. A most recent, excellent, more comprehensive review article on the topic of root hair and trichome development. 11. Marks MD: Molecular genetic analysis of trichome development in Arabidopsis. Annu Rev Plant Physiol Plant Mol Biol 1997, 48:137-163. 12. Balkunde R, Pesch M, Hu¨lskamp M: Trichome patterning in Arabidopsis thaliana from genetic to molecular models. Curr Top Dev Biol 2010, 91:299-321. A recent, more comprehensive review article on the topic of trichome patterning. 13. Pesch M, Hu¨lskamp M: One, two, three . . . models for trichome patterning in Arabidopsis? Curr Opin Plant Biol 2009, 12:587-592. 14. Rerie WG, Feldmann KA, Marks MD: The GLABRA2 gene encodes a homeo domain protein required for normal trichome development in Arabidopsis. Genes Dev 1994, 8:1388-1399. 15. Walker AR, Davison PA, Bolognesi-Winfield AC, James CM, Srinivasan N, Blundell TL, Esch JJ, Marks MD, Gray JC: The TRANSPARENT TESTA GLABRA1 locus, which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis, encodes a WD40 repeat protein. Plant Cell 1999, 11:1337-1350. 16. Oppenheimer DG, Herman PL, Sivakumaran S, Esch J, Marks MD: A myb gene required for leaf trichome differentiation in Arabidopsis is expressed in stipules. Cell 1991, 67:483-493. 17. Kirik V, Lee MM, Wester K, Herrmann U, Zheng Z, Oppenheimer D, Schiefelbein J, Hu¨lskamp M: Functional diversification of MYB23 and GL1 genes in trichome morphogenesis and initiation. Development 2005, 132:1477-1485. 18. Payne CT, Zhang F, Lloyd AM: GL3 encodes a bHLH protein that regulates trichome development in Arabidopsis through interaction with GL1 and TTG1. Genetics 2000, 156:1349-1362. 19. Bernhardt C, Lee MM, Gonzalez A, Zhang F, Lloyd A, Schiefelbein J: The bHLH genes GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) specify epidermal cell fate in the Arabidopsis root. Development 2003, 130:6431-6439. 20. Zhang F, Gonzalez A, Zhao M, Payne CT, Lloyd A: A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis. Development 2003, 130:4859-4869. Current Opinion in Plant Biology 2012, 15:31–37
21. Lee MM, Schiefelbein J: WEREWOLF, a MYB-related protein in Arabidopsis, is a position-dependent regulator of epidermal cell patterning. Cell 1999, 99:473-483.
25. Kirik V, Simon M, Wester K, Schiefelbein J, Hu¨lskamp M: ENHANCER of TRY and CPC 2 (ETC2) reveals redundancy in the region-specific control of trichome development of Arabidopsis. Plant Mol Biol 2004, 55:389-398. 26. Tominaga R, Iwata M, Sano R, Inoue K, Okada K: Arabidopsis C, APRICE-LIKE MYB 3 (CPL3) controls endoreduplication and flowering development in addition to trichome and root hair formation. Development 2008, 135:1335-1345. 27. Wang S, Kwak SH, Zeng Q, Ellis BE, Chen XY, Schiefelbein J, Chen JG: TRICHOMELESS1 regulates trichome patterning by suppressing GLABRA1 in Arabidopsis. Development 2007, 134:3873-3882. 28. Wang S, Hubbard L, Chang Y, Guo J, Schiefelbein J, Chen JG: Comprehensive analysis of single-repeat R3 MYB proteins in epidermal cell patterning and their transcriptional regulation in Arabidopsis. BMC Plant Biol 2008, 8:81. 29. Wester K, Digiuni S, Geier F, Timmer J, Fleck C, Hu¨lskamp M: Functional diversity of R3 single-repeat genes in trichome development. Development 2009, 136:1487-1496. 30. Szymanski DB, Jilk RA, Pollock SM, Marks MD: Control of GL2 expression in Arabidopsis leaves and trichomes. Development 1998, 125:1161-1171. 31. Wang S, Chen JG: Arabidopsis transient expression analysis reveals that activation of GLABRA2 may require concurrent binding of GLABRA1 and GLABRA3 to the promoter of GLABRA2. Plant Cell Physiol 2008, 49:1792-1804. 32. Zhao M, Morohashi K, Hatlestad G, Grotewold E, Lloyd A: The TTG1–bHLH–MYB complex controls trichome cell fate and patterning through direct targeting of regulatory loci. Development 2008, 135:1991-1999. 33. Morohashi K, Grotewold E: A systems approach reveals regulatory circuitry for Arabidopsis trichome initiation by the GL3 and GL1 selectors. PLoS Genet 2009, 5:e1000396. 34. Savage NS, Walker T, Wieckowski Y, Schiefelbein J, Dolan L, Monk NA: A mutual support mechanism through intercellular movement of CAPRICE and GLABRA3 can pattern the Arabidopsis root epidermis. PLoS Biol 2008, 6:e235. 35. Bouyer D, Geier F, Kragler F, Schnittger A, Pesch M, Wester K, Balkunde R, Timmer J, Fleck C, Hu¨lskamp M: Two-dimensional patterning by a trapping/depletion mechanism: the role of TTG1 and GL3 in Arabidopsis trichome formation. PLoS Biol 2008, 6:e141. 36. Digiuni S, Schellmann S, Geier F, Greese B, Pesch M, Wester K, Dartan B, Mach V, Srinivas BP, Timmer J et al.: A competitive complex formation mechanism underlies trichome patterning on Arabidopsis leaves. Mol Syst Biol 2008, 4:217. 37. Benı´tez M, Alvarez-Buylla ER: Dynamic-module redundancy confers robustness to the gene regulatory network involved in hair patterning of Arabidopsis epidermis. Biosystems 2010, 102:11-15. 38. Benı´tez M, Espinosa-Soto C, Padilla-Longoria P, AlvarezBuylla ER: Interlinked nonlinear subnetworks underlie the formation of robust cellular patterns in Arabidopsis epidermis: a dynamic spatial model. BMC Syst Biol 2008, 2:98. www.sciencedirect.com
The patterning of epidermal hairs in Arabidopsis — updated Grebe 37
39. Hu¨lskamp M, Misera S, Ju¨rgens G: Genetic dissection of trichome cell development in Arabidopsis. Cell 1994, 76:555-566. 40. Bramsiepe J, Wester K, Weinl C, Roodbarkelari F, Kasili R, Larkin JC, Hu¨lskamp M, Schnittger A: Endoreplication controls cell fate maintenance. PLoS Genet 2010, 6:e1000996. This is the first study directed to test for and reveal a connection between endoreplication and cell-fate maintenance based on developmental stage-specific modulation of the endoreplication machinery employing overexpression and downregulation of cell cycle regulators. 41. Churchman ML, Brown ML, Kato N, Kirik V, Hu¨lskamp M, Inze´ D, De Veylder L, Walker JD, Zheng Z, Oppenheimer DG et al.: SIAMESE, a plant-specific cell cycle regulator, controls endoreplication onset in Arabidopsis thaliana. Plant Cell 2006, 18:3145-3157. 42. Jakoby MJ, Falkenhan D, Mader MT, Brininstool G, Wischnitzki E, Platz N, Hudson A, Hu¨lskamp M, Larkin J, Schnittger A: Transcriptional profiling of mature Arabidopsis trichomes reveals that NOECK encodes the MIXTA-like transcriptional regulator MYB106. Plant Physiol 2008, 148:1583-1602. 43. Schnittger A, Scho¨binger U, Bouyer D, Weinl C, Stierhof YD, Hu¨lskamp M: Ectopic D-type cyclin expression induces not only DNA replication but also cell division in Arabidopsis trichomes. Proc Natl Acad Sci U S A 2002, 99:6410-6415. 44. Schnittger A, Weinl C, Bouyer D, Scho¨binger U, Hu¨lskamp M: Misexpression of the cyclin-dependent kinase inhibitor ICK1/ KRP1 in single-celled Arabidopsis trichomes reduces endoreduplication and cell size and induces cell death. Plant Cell 2003, 15:303-315. 45. Kasili R, Walker JD, Simmons LA, Zhou J, De Veylder L, Larkin JC: SIAMESE cooperates with the CDH1-like protein CCS52A1 to establish endoreplication in Arabidopsis thaliana trichomes. Genetics 2010, 185:257-268. See annotation to Ref. [46]. 46. Larson-Rabin Z, Li Z, Masson PH, Day CD: FZR2/CCS52A1 expression is a determinant of endoreduplication and cell expansion in Arabidopsis. Plant Physiol 2009, 149:874-884. This paper along with Ref. [45] do not address pattern formation but neatly establish the role of CCS52A1 in endoreplication by loss-of-function and gain-of-function studies [45,46] and especially also in its interaction with SIM through double-mutant analysis [46] which also aids the understanding of CCS52A1 and SIM function in trichome fate maintenance [40]. 47. Desvoyes B, Ramirez-Parra E, Xie Q, Chua NH, Gutierrez C: Cell type-specific role of the retinoblastoma/E2F pathway during Arabidopsis leaf development. Plant Physiol 2006, 140:67-80. 48. Wenger JP, Marks MD: E2F and retinoblastoma related proteins may regulate GL1 expression in developing Arabidopsis trichomes. Plant Signal Behav 2008, 3:420-422. 49. Wang S, Barron C, Schiefelbein J, Chen JG: Distinct relationships between GLABRA2 and single-repeat R3 MYB transcription factors in the regulation of trichome and root hair patterning in Arabidopsis. New Phytol 2010, 185:387-400. This study employs multiple mutant analyses which reveals unexpected interactions between GL2 and the single-repeat R3 MYB transcriptional regulators suggesting that they act through factors in addition to GL2 which now need to be discovered.
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50. Kawagoe T, Shimizu KK, Kakutani T, Kudoh H: Coexistence of trichome variation in a natural plant population: a combined study using ecological and candidate gene approaches. PLoS One 2011, 6:e22184. 51. Hilscher J, Schlo¨tterer C, Hauser MT: A single amino acid replacement in ETC2 shapes trichome patterning in natural Arabidopsis populations. Curr Biol 2009, 9:1747-1751. This work based on QTL mapping and subsequent comparison of a large number of ETC2 alleles from natural populations reveals new mutations in a previously uncharacterized domain of ETC2 that enhance or reduce its repressor activity. It demonstrates the power of analyses of natural allelic variation both for our understanding of molecular function and of the contribution of loci to patterning in natural populations. 52. Symonds VV, Hatlestad G, Lloyd AM: Natural allelic variation defines a role for ATMYC1: trichome cell fate determination. PLoS Genet 2011, 7:e1002069. This study based on QTL mapping and subsequent comparison of a large number of alleles from natural populations identifies AtMYC1 as a novel regulator of trichome patterning in Arabidopsis. The causative SNP discovered at the AtMYC1 locus from several low-trichome-density accessions also abolishes the protein interactions of AtMYC1 with GL1 and TTG1 discovered in the yeast two-hybrid system. The work reveals the first known function of AtMYC1. 53. Kwak SH, Schiefelbein J: A feedback mechanism controlling SCRAMBLED receptor accumulation and cell-type pattern in Arabidopsis. Curr Biol 2008, 18:1949-1954. 54. Hassan H, Scheres B, Blilou I: JACKDAW controls epidermal patterning in the Arabidopsis root meristem through a noncell-autonomous mechanism. Development 2010, 137:1523-1529. This report identifies the zinc finger protein JKD as one of the first two factors contributing positional information to root epidermal hair patterning from the underlying cortical cell layer, and it suggests that JKD acts in one pathway with the leucine-rich receptor kinase-like protein SCM. 55. Welch D, Hassan H, Blilou I, Immink R, Heidstra R, Scheres B: Arabidopsis JACKDAW and MAGPIE zinc finger proteins delimit asymmetric cell division and stabilize tissue boundaries by restricting SHORT-ROOT action. Genes Dev 2007, 21:2196-2204. 56. Mylona P, Linstead P, Martienssen R, Dolan L: SCHIZORIZA controls an asymmetric cell division and restricts epidermal identity in the Arabidopsis root. Development 2002, 129:4327-4334. 57. ten Hove CA, Willemsen V, de Vries WJ, van Dijken A, Scheres B, Heidstra R: SCHIZORIZA encodes a nuclear factor regulating asymmetry of stem cell divisions in the Arabidopsis root. Curr Biol 2010, 20:452-457. See annotation to Ref. [58]. 58. Pernas M, Ryan E, Dolan L: SCHIZORIZA controls tissue system complexity in plants. Curr Biol 2010, 20:818-823. This paper along with Ref. [57] independently identifies a role for the SCZ protein in cell-fate separation and Ref. [57] further demonstrates that SCZ acts non-cell autonomously on root epidermal patterning. These are also the first studies to identify a role for a HEAT SHOCK TRANSCRIPTION FACTOR B4 in cell-fate separation and patterning. They open new horizons for our understanding of specific functions of such proteins during development.
Current Opinion in Plant Biology 2012, 15:31–37