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Comprehensive analysis of single-repeat R3 MYB proteins in epidermal cell patterning and their transcriptional… Wang, Shucai; Hubbard, Leah; Chang, Ying; Guo, Jianjun; Schiefelbein, John; Chen, Jin-Gui Jul 21, 2008

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ralssBioMed CentBMC Plant BiologyOpen AcceResearch articleComprehensive analysis of single-repeat R3 MYB proteins in epidermal cell patterning and their transcriptional regulation in ArabidopsisShucai Wang1, Leah Hubbard2, Ying Chang1, Jianjun Guo1, John Schiefelbein*2 and Jin-Gui Chen*1Address: 1Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada and 2Department of Molecular, Cell, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USAEmail: Shucai Wang - shucaiw@interchange.ubc.ca; Leah Hubbard - hubbardl@umich.edu; Ying Chang - yingchang1970@yahoo.com; Jianjun Guo - jimguo@interchange.ubc.ca; John Schiefelbein* - schiefel@umich.edu; Jin-Gui Chen* - jingui@interchange.ubc.ca* Corresponding authors    AbstractBackground: Single-repeat R3 MYB transcription factors are critical components of the lateralinhibition machinery that mediates epidermal cell patterning in plants. Sequence analysis of theArabidopsis genome using the BLAST program reveals that there are a total of six genes, includingTRIPTYCHON (TRY), CAPRICE (CPC), TRICHOMELESS1 (TCL1), and ENHANCER of TRY and CPC 1, 2,and 3 (ETC1, ETC2 and ETC3) encoding single-repeat R3 MYB transcription factors that areapproximately 50% identical to one another at the amino acid level. Previous studies indicate thatthese single-repeat R3 MYBs regulate epidermal cell patterning. However, each of the previousstudies of these single-repeat R3 MYBs has been limited to an analysis of only a subset of these sixgenes, and furthermore, they have limited their attention to epidermal development in only one ortwo of the organs. In addition, the transcriptional regulation of these single-repeat R3 MYB genesremains largely unknown.Results: By analyzing multiple mutant lines, we report here that TCL1 functions redundantly withother single-repeat R3 MYB transcription factors to control both leaf trichome and root hairformation. On the other hand, ETC1 and ETC3 participate in controlling trichome formation oninflorescence stems and pedicles. Further, we discovered that single-repeat R3 MYBs suppresstrichome formation on cotyledons and siliques, organs that normally do not bear any trichomes.By using Arabidopsis protoplast transfection assays, we found that all single-repeat R3 MYBsexamined interact with GL3, and that GL1 or WER and GL3 or EGL3 are required and sufficientto activate the transcription of TRY, CPC, ETC1 and ETC3, but not TCL1 and ETC2. Furthermore,only ETC1's transcription was greatly reduced in the gl3 egl3 double mutants.Conclusion: Our comprehensive analysis enables us to draw broader conclusions about the roleof single-repeat R3 MYB gene family than were possible in the earlier studies, and reveals thegenetic basis of organ-specific control of trichome formation. Our findings imply the presence ofmultiple mechanisms regulating the transcription of single-repeat R3 MYB genes, and provide newPublished: 21 July 2008BMC Plant Biology 2008, 8:81 doi:10.1186/1471-2229-8-81Received: 27 May 2008Accepted: 21 July 2008This article is available from: http://www.biomedcentral.com/1471-2229/8/81© 2008 Wang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 13(page number not for citation purposes)insight into the lateral inhibition mechanism that mediates epidermal cell patterning.BMC Plant Biology 2008, 8:81 http://www.biomedcentral.com/1471-2229/8/81BackgroundThere are a total of six genes in the Arabidopsis genomethat encode a unique subfamily of MYB transcription fac-tors, namely single-repeat R3 MYB transcription factors.These transcription factors, including TRIPTYCHON(TRY) [1,2], CAPRICE (CPC) [3], TRICHOMELESS1(TCL1) [4], ENHANCER of TRY and CPC 1, 2, and 3(ETC1, ETC2 and ETC3 (CPL3)) [5-9], are characterizedby their short sequence (75–112 amino acids) and consistlargely of the single MYB domain (e.g. without other pre-dicted motifs). It is generally believed that these single-repeat R3 MYB transcription factors mediate lateral inhi-bition during epidermal patterning. In general, these sin-gle-repeat R3 MYB transcription factors act as negativeregulators for trichome formation in shoots, but as posi-tive regulators for root hair formation in roots.T-DNA insertion mutants are available for each of thesesix single-repeat R3 MYB genes. Among them, only singleloss-of-function mutants for TRY, CPC and TCL1 displaymajor defects in trichome and/or root hair cell specifica-tion [1-4], whereas loss-of-function alleles of ETC1, ETC2or ETC3 cause little or no phenotypic effect [5-9]. Theanalysis of double and triple mutants indicated that ETC1and ETC3 can function redundantly with TRY and CPC tocontrol leaf trichome and root hair formation [5,6,9], andthat ETC2 functions redundantly with TRY and CPC tocontrol trichome formation on petioles [7]. Further, CPCfunctions redundantly with TCL1 to control trichome for-mation on the inflorescence stems and pedicels [4]. How-ever, a role of TCL1 in leaf trichome and root hairformation has not been established.Available evidence suggests that single-repeat R3 MYBtranscription factors, a WD40-repeat protein, TRANSPAR-ENT TESTA GLABRA1 (TTG1) [10,11], an R2R3 MYB-typetranscription factor, GLABRA1 (GL1) [12] or WEREWOLF(WER) [13-15], a bHLH transcription factor, GLABRA3(GL3) or ENHANCER OF GLABRA3 (EGL3) [16,17], anda homeodomain protein, GLABRA2 (GL2) [18,19], regu-late trichome and/or root hair formation (reviewed in[20-22]). Based on the results of yeast-two-hybrid interac-tion assays, it has been proposed that TTG1, GL1 or WER,and GL3 or EGL3 form an activator complex to induceGL2 expression [16]. The single-repeat R3 MYB transcrip-tion factors are proposed to move from a trichome precur-sor cell to its neighboring cell (in the shoot epidermis) orfrom an N cell to an H cell (in the root epidermis) to com-pete with GL1 or WER for binding GL3 or EGL3, thus lim-iting the activity of the activator complex [2,3,20-24].Recently, we showed that one of the single-repeat R3 MYBtranscription factors, TCL1, can directly suppress the tran-scription of GL1 [4], providing an additional loop of reg-It has been proposed that the same activator complex thatactivates GL2 can also activate the expression of single-repeat R3 MYB genes (reviewed in [20-22]), but so faronly CPC has been identified as a direct target gene forWER [25,26], and GL3 has been shown to be recruited tothe promoter region of CPC and ETC1 [27].To gain new insight into the role of single-repeat R3 MYBtranscription factors in controlling epidermal develop-ment, we conduct a comprehensive analysis of the single-repeat R3 MYB gene family. By generating and analyzinghigher order mutants among six single-repeat R3 MYBgenes, we identified previously unrecognized roles of sin-gle-repeat R3 MYB transcription factors in the regulationof trichome and root hair formation. We demonstrate thatTCL1 participates in the control of leaf trichome and roothair formation, and that ETC1 and ETC3 have a role inregulating trichome formation on the inflorescence stemsand pedicles. We also discover that single-repeat R3 MYBsnormally suppress trichome formation on siliques andcotyledons. By using an Arabidopsis protoplast transfec-tion system, we show that cotransfection of GL1 or WER,with GL3 or EGL3, is sufficient to activate the transcrip-tion of TRY, CPC, ETC1 and ETC3, but not ETC2 andTCL1. Our results suggest that although the six single-repeat R3 MYB genes have largely overlapping functionsin controlling epidermal development, the transcriptionalregulation of these single-repeat R3 MYB genes involvesdistinct mechanisms.ResultsSingle-repeat R3 MYB transcription factors in ArabidopsisSequence analysis of the Arabidopsis genome using theBLAST program http://www.ncbi.nlm.nih.gov/ with theentire amino acid sequence of TCL1 reveals that there area total of six genes, including TRY, CPC, TCL1, ETC1,ETC2 and ETC3, encoding single-repeat R3 MYB transcrip-tion factors that are approximately 50% identical to oneanother at the amino acid level. These six genes are une-venly distributed in four of the five chromosomes of Ara-bidopsis (Figure 1A). Among them, ETC2 (At2g30420)and TCL1 (At2g30432) are tandem genes located in chro-mosome II (Figure 1A). The amino acid signature [D/E]L× 2 [R/K] × 3L × 6L × 3R [28] that has been shown to berequired for interacting with R/B-like bHLH transcriptionfactors is completely conserved in all single-repeat R3MYB transcription factors (Figure 1B). The amino acidswithin the MYB domain that have been shown to be cru-cial for the cell-to-cell movement of CPC [29] are alsoentirely conserved in all single-repeat R3 MYBs (Figure1B). Phylogenetic analysis using the entire amino acidsequence suggested that these six single-repeat R3 MYBtranscription factors can be divided into two groups, withPage 2 of 13(page number not for citation purposes)ulation of the activity of the proposed activator complexby single-repeat R3 MYB transcription factors.TCL1, ETC1, ECT3 and CPC in group I, and TRY and ETC2in group II (Figure 1C).BMC Plant Biology 2008, 8:81 http://www.biomedcentral.com/1471-2229/8/81TCL1 affects both leaf trichome and root hair formationAmong the six single-repeat R3 MYB transcription factors,TRY and CPC have been shown to be involved in both leaftrichome and root hair formation [1-3,30], while ETC1can function redundantly with TRY and CPC to controlleaf trichome and root hair formation [5,6] and ETC2functions redundantly with TRY and CPC to control tri-chome formation on petioles [7]. Recently, it has beenshown that ETC3 functions redundantly with single-repeat R3 MYBs to regulate both trichome and root hairformation [9]. However, a role of TCL1 in leaf trichomeand root hair formation has not been established,[4]. To test if TCL1 participates in the control of leaf tri-chome and root hair formation, we generated double, tri-ple and quadruple mutants among all single-repeat R3MYB genes in group I including TCL1, ETC1, ETC3 andCPC (Figure 1C). Consistent with previous report [2], thecpc single mutant has significantly increased trichomenumber on leaves (Figure 2A, Table 1). All other singlemutants of group I genes examined are indistinguishablefrom wild-type plants (Table 1). We found that althoughdouble and triple mutants including cpc (e.g. cpc etc1 etc3)are similar to the cpc single mutant, and double and triplemutants between etc1, etc3, and tcl1 are similar to wildSingle-repeat R3 MYB transcription factors in ArabidopsisFigure 1Single-repeat R3 MYB transcription factors in Arabidopsis. (A) Chromosome location of six single-repeat R3 MYB genes. (B) Amino acid sequence alignment of single-repeat R3 MYB transcription factors. Identical amino acids are shaded in black and similar amino acids in gray. The single R3 MYB domain is indicated with think lines underneath. The amino acid signa-ture [D/E]L × 2 [R/K] × 3L × 6L × 3R [28] that is required for interacting with R/B-like BHLH transcription factors is indicated by arrowheads on the top of amino acids. The amino acids within the MYB domain that are crucial for cell-to-cell movement of CPC [29] are indicated by asterisks on the top of amino acids. (C) Phylogenetic analysis of the single-repeat R3 MYB transcrip-tion factors. The phylogenetic tree using the entire amino acid sequence was generated using software AliBee – Multiple align-ment Release 2.0 http://www.genebee.msu.su/services/malign_reduced.htmlPage 3 of 13(page number not for citation purposes)although previously, we showed that TCL1 controls tri-chome formation on the inflorescence stems and pediclestype (Table 1), the cpc etc1 etc3 tcl1 quadruple mutant hasdramatically increased trichome number on leaves thanBMC Plant Biology 2008, 8:81 http://www.biomedcentral.com/1471-2229/8/81cpc single mutant and cpc etc1 etc3 triple mutant (Figure2A, Table 1), indicating that TCL1 is involved in the regu-lation of leaf trichome formation.Subsequently, we examined root hair formation in thesemutants. As shown in Figure 2B and Table 2, cpc singlemutant had reduced root hair formation, consistent withprevious reports [3,30]. Compared with the cpc singlemutant, the cpc etc1 etc3 triple mutant produced fewerroot hairs (Figure 2B, Table 2). Moreover, cpc etc1 etc3 tcl1quadruple mutant produced significantly fewer root hairsthan cpc etc1 etc3 triple mutant (Figure 2B, Table 2), indi-cating that TCL1 also participates in the regulation of roothair formation.ETC1 and ETC3 participate in the control of trichome formation on the inflorescence stems and pediclesPreviously, we reported that TCL1 is a major regulator oftrichome formation on the inflorescence stem and pedi-cels because tcl1 single mutants displayed ectopic tri-chome formation on pedicels, and trichome formation onof the single-repeat R3 MYB gene family displayed ectopictrichome formation on the inflorescence stems or pedi-cels. We have previously shown that CPC acts synergisti-cally with TCL1 to control trichome formation on theseorgans [4]. In this study, we wanted to further investigateif any other single-repeat R3 MYBs can also regulate tri-chome formation on the inflorescence stems and pedicels.We examined trichome formation on the inflorescencestems and pedicles in the double, triple and quadruplemutants of all single-repeat R3 MYB genes in group Iincluding TCL1, ETC1, ETC3 and CPC. Compared withthe tcl1 single mutant or the cpc tcl1 double mutant, cpcetc1 etc3 tcl1 quadruple mutants have more internodesbearing trichomes (Figure 3A) and more pedicels formingectopic trichomes (Figure 3B). In addition, ectopic tri-chome formation was also found on the pedicels of thecpc etc1 etc3 triple mutants (Figure 3B, Figure 4A). Unlikethe tcl1 mutant, whose trichomes are evenly distributedalong pedicels, trichomes tend to form at both ends of thepedicels of the cpc etc1 etc3 mutant (Figure 4A). TheseTable 1: Leaf trichome production in wild-type, mutants, and transgenic Lines. Values indicate mean ± standard deviation of at least ten rosette leaves for each line.Genotype Number of Trichomes Per Leaf Frequency of Trichome Clusters (%)WT (Col) 33.5 ± 6.0 0WT (WS) 31.9 ± 5.7 0cpc-1 56.5 ± 10.2 * 0try-29760 38.4 ± 7.2 12.1 *tcl1-1 26.8 ± 5.1 0etc1-1 40.8 ± 7.2 0etc2-2 32.7 ± 4.2 0etc3-1 31.3 ± 4.0 0etc3-1 cpc-1 59.5 ± 6.6 * 1.2 *etc3-1 try-29760 31.3 ± 4.2 8.8 *etc3-1 tcl-1 33.9 ± 5.0 0.3etc3-1 etc1-1 28.8 ± 4.2 0.5etc3-1 etc2-2 35.5 ± 6.1 0tcl1-1 cpc-1 49.9 ± 8.5 * 2.4 *tcl1-1 try-29760 32.5 ± 6.3 9.5 *cpc-1 try-29760 156 ± 28 * 89 *cpc-1 try-29760 tcl1-1 135 ± 36 * 93 *cpc-1 try-29760 etc1-1 190 ± 42 * 99 *cpc-1 tcl1-1 etc3-1 46.6 ± 7.8 * 1.4 *cpc-1 etc1-1 etc3-1 51.9 ± 8.7 * 7.0 *cpc-1 tcl1-1 etc1-1 40.5 ± 3.2 0.4etc3-1 try-29760 tcl1-1 31.6 ± 4.5 11.5 *cpc-1 tcl1-1 etc1-1 etc3-1 106 ± 23 *# 16 *cpc-1 try-29760 tcl1-1 etc1-1 182 ± 38 * 98 *35S:HA-ETC3 0 ± 0 * 0PETC3:ETC3-GFP 0 ± 0 * 0* p < 0.05, relative to the corresponding wild type line.# p < 0.05, relative to the cpc-1 etc1-1 etc3-1 line.Page 4 of 13(page number not for citation purposes)inflorescence stems was no longer restricted to the inter-nodes before the first flower [4]. No other single mutantresults supported the notion that TCL1 is the major regu-lator of the single-repeat R3 MYB family controlling tri-BMC Plant Biology 2008, 8:81 http://www.biomedcentral.com/1471-2229/8/81chome formation on the inflorescence stems and pedicels,and that CPC, ETC1 and ETC3 participate in the control oftrichome formation on those organs.Single-repeat R3 MYBs function redundantly to control trichome formation on siliques and cotyledonsHaving generated quadruple mutants with combinationof loss-of-function mutations in all group I members ofsingle-repeat R3 MYB genes, including CPC, ETC1, TCL1and ETC3, we sought to generate higher order mutants.The group II small MYBs contains two members, TRY andETC2 (Figure 1C). As discussed above, ETC2 (At2g30420)and TCL1 (At2g30432) are tandem genes located in thechromosome II (Figure 1A), which inhibits the generationof sextuple mutants that contain loss-of-function muta-tions in all six single-repeat R3 MYB genes through classi-cal crosses. Therefore, in this study, the highest ordermutant we generated was a quintuple mutant containsloss-of-function mutations in all four members of group Iand TRY of group II of the single-repeat R3 MYB gene fam-ily.By analyzing the try cpc etc1 etc3 tcl1 quintuple mutant andformed on the siliques (Figure 4B). Second, ectopic tri-chomes were formed on the cotyledons of try cpc etc1 etc3tcl1 quintuple mutant (Figure 4C). Because these organsin wild-type plants do not bear any trichomes, theseresults indicated that single-repeat R3 MYB transcriptionfactors normally suppress trichome formation on theseorgans and acts in a highly redundant manner.Single-repeat R3 MYBs function redundantly to control trichome cluster formation on leaves and inflorescence stemsAmong all single mutants of the single-repeat R3 MYBgenes, only the try mutant displays significant trichomeclusters on leaves and shoots [1,2]. We wanted to examinewhether other single-repeat R3 MYB transcription factorsfunction redundantly with TRY to control trichome spac-ing. Consistent with that reported previously [6], the sizeof the trichome clusters on leaves increased significantlyfrom the try cpc double mutant to the try cpc etc1 triplemutant (Figure 5A). We found that the size of trichomeclusters on leaves was further increased in the try cpc etcltcl1 quadruple mutant (Figure 5A), suggesting that TCL1also contributes to the control of trichome spacing onleaves.We also found that etc3-1 mutation can increase trichomecluster frequency in cpc mutant background. As shown inTable 1, the cpc, etc1, tcl1, and etc3 single mutants are sim-ilar to wild type in term of trichome cluster formation.However, in the cpc etc3 double mutant, about 1.2% of itstrichomes are in clusters, and the number of trichomeclusters was increased to about 7% in cpc etc1 etc3, andabout 16% in cpc etc1 etc3 tcl1 (Table 1). These results sup-ported the notion that TRY is the major regulator for con-trolling trichome cluster formation, and suggested thatother single-repeat R3 MYBs contribute to trichome clus-ter formation by functioning in a redundant manner.Subsequently, we examined trichome cluster formationon the inflorescence stems. As discussed earlier, TCL1 is amajor regulator of trichome formation on the inflores-cence stems whereas TRY controls the formation of tri-chome cluster. As expected, extensive trichome clusterswere found on the inflorescence stems of try cpc etc1 tcl1quadruple mutants (Figure 5B).Single-repeat R3 MYB transcription factors interact with GL3 in plant cellsOur genetic analyses indicated that single-repeat R3 MYBsregulate trichome and root hair formation in a highlyredundant manner. We next wished to study their mecha-nism of action in trichome and root hair formation. It hasbeen shown that some of these single-repeat R3 MYB tran-TCL1 participates in the control of both leaf trichome and root hair formationFigure 2TCL1 participates in the control of both leaf tri-chome and root hair formation. (A) TCL1 functions redundantly with other single-repeat R3 MYB transcription factors to control trichome formation on leaves. Pictures were taken from 2-week-old, soil-grown seedlings. (B) TCL1 functions redundantly with other single-repeat R3 MYB tran-scription factors to control root hair formation. Pictures were taken from 7-day-old seedlings grown on vertically ori-entated 1/2 MS plates.Page 5 of 13(page number not for citation purposes)other mutants, we made two discoveries. First, in try cpcetc1 tcl1 quadruple mutants, ectopic trichomes werescription factors, including TRY, CPC, ETC1, ETC2 andETC3, interact with GL3 in yeast cells [7,9,17,24,28]. ThisBMC Plant Biology 2008, 8:81 http://www.biomedcentral.com/1471-2229/8/81property has been proposed to enable the single-repeat R3MYB transcription factors to compete with GL1 for bind-ing GL3, thus limiting the activity of TTG1-GL3/EGL3-GL1/WER activator complex and inhibiting trichome for-mation and promoting root hair formation [20-23].Although TCL1 is likely to behave similarly as TRY, CPC,ETC1, ETC2 and ETC3, a direct test of the interactionsbetween TCL1 and GL3 has not been performed. Further,the interactions in yeast cells (e.g. interaction betweenGL3 and TRY, CPC, ETC1, ETC2 or ETC3) have not beenconfirmed in plant cells. Therefore, in this study, we testedthe interactions between GL3 and each of these six single-repeat R3 MYB transcription factors using a plant two-hybrid protein-protein interaction system [31].A Gal4-GUS reporter, together with the effectors GL3 anda Gal4 DNA binding domain (GD) fused single-repeat R3MYB transcription factor (Figure 6A), were co-transfectedinto Arabidopsis protoplasts. Although single-repeat R3MYB transcription factors can be recruited to the promoterregion of the reporter gene by GD, they alone cannot acti-expression can occur if the small MYB protein interactswith GL3, because GL3 can function as a transcriptionalactivator (data not shown). Indeed, TRY, CPC, ETC1,ETC2 and ETC3 interacted with GL3 in plant cells (Figure6B), consistent with the results reported in yeast cells[7,9,17,24,28]. Using this system, we also showed thatTCL1 is able to interact with GL3 (Figure 6B). Thus, all sixsingle-repeat R3 MYB transcription factors can interactwith GL3 in plant cells, implying that GL3 binding mayrepresent a general mechanism of single-repeat R3 MYBaction in inhibiting the activity of the TTG1-GL3/EGL3-GL1 activator complex.Transcriptional regulation of single-repeat R3 MYB genesIt has been proposed that the TTG1-GL3/EGL3-GL1 acti-vator complex in shoots not only promotes the transcrip-tion of GL2, but also promotes the transcription of single-repeat R3 MYB genes, and a similar mechanism has beenproposed to operate in roots with WER replacing GL1(reviewed in [20-22]). In support of this view, GL3 hasbeen shown to be recruited to the promoter region of CPCTable 2: Root-hair and non-hair cell specification in the root epidermis of wild-type, mutant, and transgenic Lines.Genotype Hair cells in epidermis (%) H cell position N cell positionHair cells (%) Non-hair cells (%) Hair cells (%) Non-hair cells (%)WT (Col) 41.6 ± 2.9 97.1 ± 1.7 2.9 ± 1.7 0.5 ± 1.1 99.5 ± 1.1WT (WS) 40.2 ± 4.2 96.2 ± 2.4 3.8 ± 2.4 0.3 ± 0.3 99.7 ± 0.3cpc-1 14.2 ± 2.4 * 24.2 ± 2.7 75.8 ± 2.7 0.5 ± 1.0 99.5 ± 1.0try-29760 40.0 ± 4.9 94.5 ± 3.5 5.5 ± 3.5 1.3 ± 2.0 98.7 ± 2.0tcl1-1 42.7 ± 4.4 95.8 ± 2.9 4.2 ± 2.9 0.2 ± 0.6 99.8 ± 0.6etc1-1 39.4 ± 3.2 93.8 ± 4.2 6.2 ± 4.2 1.8 ± 1.9 98.2 ± 1.9etc2-2 38.5 ± 5.4 95.9 ± 4.9 4.1 ± 4.9 0 ± 0 100 ± 0etc3-1 39.0 ± 4.1 96.7 ± 3.5 3.3 ± 3.5 1.1 ± 0.6 98.9 ± 0.6etc3-1 cpc-1 16.6 ± 3.3* 27.8 ± 4.2 72.2 ± 4.2 0.5 ± 0.5 99.5 ± 0.5etc3-1 try-29760 39.1 ± 4.9 95.0 ± 3.8 5.0 ± 3.8 1.9 ± 2.1 98.1 ± 2.1etc3-1 tcl-1 41.8 ± 3.0 96.7 ± 2.9 3.3 ± 2.9 1.1 ± 1.0 98.9 ± 1.0etc3-1 etc1-1 40.1 ± 4.3 94.1 ± 5.3 5.9 ± 5.3 1.8 ± 2.5 98.2 ± 2.5etc3-1 etc2-2 38.5 ± 5.5 96.0 ± 3.9 4.0 ± 3.9 0.6 ± 1.0 99.4 ± 1.0tcl1-1 cpc-1 14.8 ± 3.0 * 24.4 ± 4.1 75.6 ± 4.1 0 ± 0 100 ± 0tcl1-1 try-29760 40.5 ± 2.9 93.9 ± 4.0 6.1 ± 4.0 1.2 ± 1.2 98.8 ± 1.2cpc-1 try-29760 0 ± 0 * 0 ± 0 100 ± 0 0 ± 0 100 ± 0cpc-1 try-29760 tcl1-1 0 ± 0 * 0 ± 0 100 ± 0 0 ± 0 100 ± 0cpc-1 try-29760 etc1-1 0 ± 0 * 0 ± 0 100 ± 0 0 ± 0 100 ± 0cpc-1 tcl1-1 etc3-1 20.9 ± 3.9 * 41.7 ± 5.8 58.3 ± 5.8 0.6 ± 1.3 99.4 ± 1.3cpc-1 etc1-1 etc3-1 8.5 ± 2.7 * 16.5 ± 4.0 83.5 ± 4.0 0 ± 0 100 ± 0cpc-1 tcl1-1 etc1-1 22.6 ± 4.1 * 43.2 ± 5.1 56.8 ± 5.1 0.2 ± 0.4 99.8 ± 0.4etc3-1 try-29760 tcl1-1 41.3 ± 4.7 95.1 ± 4.4 4.9 ± 4.4 0.9 ± 1.2 99.1 ± 1.2cpc-1 tcl1-1 etc1-1 etc3-1 3.4 ± 1.9 *# 9.2 ± 2.6 90.8 ± 2.6 0 ± 0 100 ± 0cpc-1 try-29760 tcl1-1 etc1-1 0 ± 0 * 0 ± 3.3 100 ± 0 0 ± 0 100 ± 035S:HA-ETC3 48.8 ± 5.2 97.7 ± 3.1 2.3 ± 3.1 15.1 ± 3.9 84.9 ± 3.9PETC3:ETC3-GFP 41.9 ± 4.4 96.1 ± 3.9 3.9 ± 3.9 2.6 ± 2.2 97.4 ± 2.2Values indicate mean ± standard deviation of at least 10 roots for each line. In all strains, approximately 40% of epidermal cells are in the H position.* p < 0.05, relative to the corresponding wild type line.# p < 0.05, relative to the cpc-1 etc1-1 etc3-1 line.Page 6 of 13(page number not for citation purposes)vate the expression of the reporter gene due to the lack ofa transcription-activating domain. Activation of reporterand ETC1 [27], and CPC has been identified as a direct tar-get gene for WER [26].BMC Plant Biology 2008, 8:81 http://www.biomedcentral.com/1471-2229/8/81To investigate whether activation of single-repeat R3 MYBgenes by the TTG1-GL3/EGL3-GL1/WER complex repre-sents a general regulatory mechanism, we used an Arabi-dopsis mesophyll protoplast transfection system [32,33].This system has been successfully used to elucidate theregulatory roles of many other transcription factors [34–38). First, we examined the expression of each componentand EGL3 transcripts were undetectable by RT-PCR inwild-type protoplasts, whereas all of them were expressedin the whole seedlings (Figure 7A). These results indicatedthat Arabidopsis rosette leaf mesophyll protoplasts aresuitable for the study of transcriptional activity of theseactivator complexes by transfection assays because thepotential interferences by the endogenous TTG1, WER,GL1, GL3 and EGL3 are minimized. We also found thatthe basal transcript level of TRY was highest among all sin-gle-repeat R3 MYB genes in untransfected protoplasts (Fig-ETC1 and ETC3 participate in the control of trichome for-mation on the inflo escence stems and pediclesFigure 3ETC1 and ETC3 participate in the control of tri-chome formation on the inflorescence stems and pedicles. (A) Single-repeat R3 MYB transcription factors function redundantly to control trichome formation on the inflorescence stems. (B) Single-repeat R3 MYB transcription factors function redundantly to control trichome formation on pedicles. Data represent the mean ± SD of at least 10 plants.Single-repeat R3 MYB transcription factors function redun-dantly to control trichome formation on pedicels, siliques and co yledonsFigure 4Single-repeat R3 MYB transcription factors function redundantly to control trichome formation on pedi-cels, siliques and cotyledons. (A) Comparison of pedicle trichomes in the tcl1 single mutant (left) and the cpc etc1 etc3 triple mutant (right). Shown are pedicles of the first flowers on the main inflorescence stems. Arrow heads indicate tri-chomes on both ends of pedicels in cpc etc1 etc3 triple mutant. (B) Ectopic trichome formation on siliques of the try cpc etc1 tcl1 quadruple mutant. No trichomes were formed in the siliques of wild-type plants. (C) Ectopic trichome for-mation on cotyledons of the try cpc etc1 etc3 tcl1 quintuple mutant. No trichomes were formed in the cotyledons of wild-type seedlings. Picture was taken from a 5-day-old, light-grown seedling.Page 7 of 13(page number not for citation purposes)of the activator complex in Arabidopsis rosette leaf meso-phyll protoplasts. We found that TTG1, WER, GL1, GL3ure 7B). In a preliminary test using Arabidopsisprotoplasts from wild-type, we found that GL1 and GL3BMC Plant Biology 2008, 8:81 http://www.biomedcentral.com/1471-2229/8/81are required and sufficient to induce the expression ofCPC (data not shown). Therefore, in order to simplify ourcotransfection assays, we decided to use protoplasts pre-pared from ttg1 mutant rosette leaves to test the activationof single-repeat R3 MYB genes by the GL3/EGL3-GL1 orGL3/EGL3-WER activator complex. To further ensure thatthe elimination of TTG1 would not impair the transcrip-tional activity of the activator complex, we compared thetranscriptional activation of single-repeat R3 MYB genesby GL1+GL3 or TTG1+GL1+GL3. We found that theGL1+GL3 combination was as effective asTTG1+GL1+GL3 to activate the transcription of the single-repeat R3 MYB genes (Figure 7B). By using this system, wefound that none of the components in the proposed acti-vator complexes alone could activate the transcription ofany single-repeat R3 MYB genes (Figure 7B). However,GL1 or WER together with GL3 or EGL3 were sufficient tocated that some, but not all, single-repeat R3 MYB genesare induced by the proposed activator complexes.To further investigate if those four single-repeat R3 MYBgenes activated by the GL1-GL3/EGL3 or WER-GL3/EGL3complex, including TRY, CPC, ETC1 and ETC3, are tightlyregulated by the activator complexes, we examined theexpression of these genes in the gl3 egl3 double mutantbackground. We reasoned that if these single-repeat R3MYB genes are solely regulated by these activator com-plexes, their expression would be dramatically reduced ingl3 egl3 double mutant due to the disruption of the activa-Single-repeat R3 MYB transcription factors function redun-dantly to control trichome spacingFigure 5Single-repeat R3 MYB transcription factors function redundantly to control trichome spacing. (A) Single-repeat R3 MYB transcription factors function redundantly to control trichome cluster formation on leaves. Pictures were taken from 2-week-old, soil-grown seedlings. (B) Single-repeat R3 MYB transcription factors function redundantly to control trichome cluster formation on inflorescence stems.Single-repeat R3 MYB transcription factors interact with GL3 in plant cellsFigure 6Single-repeat R3 MYB transcription factors interact with GL3 in plant cells. (A) Effector and reporter con-structs used in transfection assays. Effector gene, Gal4 DNA binding domain (GD) fused single-repeat R3 MYB and GL3, and reporter gene, Gal4-GUS, were co-transfected into pro-toplasts derived from Arabidopsis rosette leaves. (B) Rela-tive GUS activity. GUS activity was measured after the transfected protoplasts had been incubated for 20–22 h in the darkness. Data represent the mean ± SD of three repli-cates.Page 8 of 13(page number not for citation purposes)activate the transcription of TRY, CPC, ETC1 and ETC3,but not TCL1 and ETC2 (Figure 7B). These results indi-tor complexes. Surprisingly, we found that among thesefour single-repeat R3 MYB genes, ETC1 was the only geneBMC Plant Biology 2008, 8:81 http://www.biomedcentral.com/1471-2229/8/81whose transcript was dramatically reduced in the gl3 egl3double mutant (Figure 7C). In order to more accuratelycompare the transcript level of TRY, CPC and ETC3between wild-type and gl3 egl3 mutant, we used quantita-tive real-time PCR to quantify the transcript level of thesegenes. The results from quantitative real-time PCR con-firmed that there is no difference in the transcript level ofTRY and ETC3 between wild-type and gl3 egl3 mutantDiscussionOverlapping functions of single-repeat R3 MYB transcription factors in regulating trichome and root hair formationThe six single-repeat R3 MYB transcription factors in Ara-bidopsis are highly similar to each other at the amino acidlevel (Figure 1B). However, among these six single-repeatR3 MYB genes, only single mutants of TRY, CPC and TCL1displayed major phenotypes in epidermal cell develop-ment [1-4], whereas single mutants of ETC1, ETC2 andETC3 are largely indistinguishable from wild-type plants[5-8]. Further, the phenotypes of try, cpc and tcl1 singlemutants are distinct. try mutant displays characteristic tri-chome clusters in leaves [1,2], cpc mutant has increasedtrichome formation on leaves and had decreased root hairformation [2,3], whereas tcl1 mutant produces ectopic tri-chomes on inflorescence stems and pedicels [4]. Thesefindings suggested that TRY is a major regulator regulatingtrichome cluster formation, CPC is a major regulator reg-ulating root hair and trichome formation, whereas TCL1is a major regulator regulating trichome formation oninflorescence stems and pedicels. Available evidence sug-gested that single-repeat R3 MYB transcription factors canalso function redundantly to regulate trichome and roothair formation [2,4-9]. Each of those previous studies ofthese single-repeat R3 MYBs has been limited to an analy-sis of only a subset of these six genes, and furthermore,they have limited their attentions to epidermal develop-ment in only one or two of these organs. Here we con-ducted a comprehensive analysis of the roles of single-repeat R3 MYB transcription factors in trichome and roothair formation. Our comprehensive analysis enables us todraw broader conclusions about the role of this gene fam-ily than were possible in the earlier studies.Mutants generated including the cpc etc1 etc3 tcl1 quadru-ple mutant containing loss-of-function mutations in allmembers of group I single-repeat R3 MYB genes, and thetry cpc etc1 etc3 tcl1 quintuple mutant. By analyzing thesesingle, double, triple, quadruple and quintuple mutants,we made several new discoveries. First, we established arole for TCL1 in controlling leaf trichome formation androot hair formation (Figure 2, Table 1, Table 2). TCL1 hasbeen shown to be a major regulator of the single-repeat R3MYB family controlling trichome formation on the inflo-rescence stems and pedicels [4], but a role of TCL in con-trolling trichome formation on leaves and root hairformation had not been previously established. Second,we established a role for ETC1 and ETC3 in controlling tri-chome formation on the inflorescence stems and pedicles(Figure 3). We note that a recent study showed that a dif-ferent allele of etc3, called cpl3-1, was shown to produceabout 80% more trichomes and 20% fewer root hairs thanThe regulation of transcription of single-repeat R3 MYB genes by TTG1-GL3/EGL3-GL1 activato  complexFigu e 7The regulation of transcription of single-repeat R3 MYB genes by TTG1-GL3/EGL3-GL1 activator com-plex. (A) Expression of TTG1, GL1, WER, GL3 and EGL3 in seedlings and protoplasts. RNA was isolated from 10-day-old seedlings or from protoplasts isolated from the rosette leaves of 3-4-week-old plants. RT-PCR was use to examine the expression of TTG1, GL1, WER, GL3 and EGL3. (B) Regu-lation of single-repeat R3 MYB genes by GL1-GL3/EGL3 and WER-GL3/EGL3. Effector gene(s) were transfected or cotransfected into protoplasts. RNA was isolated from trans-fected protoplasts that had been incubated for 20–22 h in the darkness. RT-PCR was used to examine the expression of single-repeat R3 MYB genes. (C) Expression of single-repeat R3 MYB genes in gl3 egl3 double mutant. RNA was isolated from 10-day-old seedlings. RT-PCR was used to examine the expression of TRY, CPC, ETC1 and ETC3. The expression of ACTIN2 was used as control.Page 9 of 13(page number not for citation purposes)whereas the transcript level of CPC was only weaklyreduced (about 20% reduction) in gl3 egl3 mutant.wild-type [9], which may be due to an allele-specific dif-ference in etc3 effect. Third, we found that mutations inBMC Plant Biology 2008, 8:81 http://www.biomedcentral.com/1471-2229/8/81TCL1, ETC1 and ETC3 can increase leaf trichome clusterfrequency in cpc mutant background (Table 1). About16% of the trichomes formed in clusters in the cpc tcl1 etc1etc3 quadruple mutant (Table 1). Working together withTRY, they also control trichome cluster formation on theinflorescence stems (Figure 5). Finally, we demonstratedthat in addition to regulating trichome formation onleaves and inflorescence stems, single-repeat R3 MYB tran-scription factors also regulate trichome formation on cot-yledons and siliques (Figure 4). Because these organsnormally do not bear any trichomes, these results sug-gested that single-repeat R3 MYBs normally suppress tri-chome formation on these organs in a highly redundantmanner.Mechanism of the action of single-repeat R3 MYB transcription factors in the regulation of trichome and root hair formationThe six single-repeat R3 MYB transcription factors areapproximately 50% identical to each other at the aminoacid level (Figure 1B). More importantly, these six single-repeat R3 MYB transcription factors contain the aminoacid signature [D/E]L × 2 [R/K] × 3L × 6L × 3R that hasbeen shown to be required for interacting with R/B-likebHLH transcription factors [28] (Figure 1B). Further, theamino acids within the MYB domain that have beenshown to be crucial for cell-to-cell movement of CPC pro-tein [29] are also entirely conserved in all six single-repeatR3 MYB transcription factors (Figure 1B). These resultsimplied that all single-repeat R3 MYB transcription factorshave the potential to act in a similar manner by movingfrom cell to cell to compete with GL1 for binding GL3,thereby limiting the activity of the TTG1-GL3/EGL3-GL1/WER activator complex to regulate trichome and/or roothair formation. Five of these six single-repeat R3 MYBtranscription factors, including TRY, CPC, ETC1, ETC2and ETC3, have been previously shown to interact withGL3 in yeast cells [7,9,17,24,28]. Here we showed that allof these six single-repeat R3 MYB transcription factorsinteract with GL3 in plant cells (Figure 6), supporting thatcompetition mechanism between single-repeat R3 MYBtranscription factors and GL1 for binding GL3 [20-23].These results suggested that single-repeat R3 MYB tran-scription factors may mediate lateral inhibition in epider-mal cell patterning by a general mechanism: competingwith GL1 for binding GL3 to reduce the activity of theTTG1-GL3/EGL3-GL1 activator complexes. It remains tobe tested if all single-repeat R3 MYB transcription factorscan also directly suppress the expression of GL1 thusinhibiting the formation of the TTG1-GL3/EGL3-GL1 acti-vate complex, as has been demonstrated for TCL1 [4].Regulation of the transcription of single-repeat R3 MYB genesPrevious studies suggested that the expression of single-repeat R3 MYB genes is regulated by the TTG1-GL3/EGL3-GL1 activator complex in shoots, and by the TTG1-GL3/EGL3-WER activator complex in roots. These single-repeatR3 MYB transcription factors move from a trichome pre-cursor cell to its neighboring cell in shoots, or move froman N cell to an H cell in roots, to compete with GL1 orWER for binding GL3, thus limiting the activity of activa-tor complexes [20-23]. Using an Arabidopsis protoplasttransfection system, we found that GL1-GL3/EGL3 orWER-GL3/EGL3 complexes are required and sufficient toactivate the expression of TRY, CPC, ETC1 and ETC3 (Fig-ure 7B). However, we did not detect the activation ofTCL1 and ETC2 by these complexes (Figure 7B). Moreo-ver, among the single-repeat R3 MYB genes that can beactivate by the GL1-GL3/EGL3 or WER-GL3/EGL3 com-plex, only the transcript of ETC1 is dramatically reducedin the gl3 egl3 double mutant (Figure 7C). These resultsimplied that: (i) only the transcription of ETC1 of thesmall MYB gene family is tightly controlled by the GL1-GL3/EGL3 and WER-GL3/EGL3 activator complexes; (ii)in addition to being controlled by the GL1-GL3/EGL3 andWER-GL3/EGL3 activator complexes, there are additionalmechanisms controlling the transcription of TRY, CPCand ETC3; and (iii) the transcription of TCL1 and ETC2are controlled by unidentified mechanisms. Therefore,our results suggested that there may be multiple, distinctmechanisms controlling the transcription of single-repeatR3 MYB genes. The differential regulation of single-repeatR3 MYB genes may contribute to the differences in theirdevelopmental functions, which might explain why somany genes with very similar function are maintained inthe genome.ConclusionWe have established that each of the single-repeat R3 MYBtranscription factors has a role in regulating trichome androot hair formation, and that these MYBs largely functionin a redundant manner. We demonstrate that a normalfunction of these MYBs is to suppress trichome formationon leaves and inflorescence stems, and to suppress tri-chome formation on cotyledons, pedicels and siliques,organs that normally do not bear any trichomes. We con-firm that GL3 binding may represent a general mecha-nism of action of single-repeat R3 MYBs in inhibiting theactivity of the TTG1-GL3/EGL3-GL1 activator complex.We show that the transcription of small MYB genes islikely regulated by multiple mechanisms. These resultsreveal genetic basis of organ-specific control of trichomeformation and provide new insight into the lateral inhibi-tion mechanism that mediates epidermal patterning.Page 10 of 13(page number not for citation purposes)BMC Plant Biology 2008, 8:81 http://www.biomedcentral.com/1471-2229/8/81MethodsPlant materials and growth conditionsThe single mutants, try_29760 [24], etc1-1 [6], tcl1-1 [4],etc2-2 and etc3-1 [8] are in the Columbia-0 (Col-0) eco-typic background. The cpc-1 mutant is in the Wassilewsk-ija (WS) ecotypic background [3]. The ttg1 and gl3 egl3mutants are in the Landsberg erecta (Ler) ecotypic back-ground [17]. Double mutants were generated by crossingsingle mutants. Triple, quadruple and quintuple mutantswere generated by crossing related lower order mutants(e.g. single, double and triple mutants). Mutants wereexamined in the F2 progeny for putative mutant pheno-type, and their mutant statuses were confirmed by geno-typing in F2 and subsequent generations. In this study, try,etc1, tcl1, etc2, etc3 and cpc refer to the specific allelestry_29760 [24], etc1-1 [6], tcl1-1 [4], etc2-2 [8], etc3-1 [8]and cpc-1 [3], respectively.Seedlings used for RT-PCR analysis were obtained bygrowing surface-sterilized seeds on 0.6% (w/v) phytoagar(plantmedia, Dublin, Ohio) solidified 1/2 Murashige &Skoog (MS) medium with vitamins (plantmedia) and 1%(w/v) sucrose. Seedling used for phenotypic analysis wereobtained either by plating seeds on 1/2 MS medium or bydirectly sowing seeds into soil. Plants were grown at 23°Cwith 14/10 h photoperiod at approximately 120 μmol m-2 s-1.Plasmid constructionConstructs used for protoplasts transfection were gener-ated by first amplifying the full-length open-readingframe (ORF) of the corresponding genes by RT-PCR usingRNA isolated from 10-d old, light-grown Arabidopsisseedlings, then cloning the PCR fragment in frame with anamino terminal HA or GD tag into the pUC19 vectorunder the control of the double 35S enhancer promoter ofCaMV [36,39]. For plant transformation, correspondingconstructs in pUC19 vector was digested with EcoRI, thensub-cloned into binary vector pPZP211 or pPZP221 [40].Plant transformation and selection of transgenic plantsFive-week-old soil grown plants with several mature flow-ers on the main inflorescence stem were used to transformwith related constructs in Agrobacterium tumefaciensGV3101 by the floral dip method [41]. Phenotypes oftransgenic plants were examined in the T1 generation, andconfirmed in T2 up to T4 generations. For all transgenicplants, at least five transgenic lines with similar pheno-types were obtained.Protoplasts isolation, transfection and GUS activity assayProtoplast isolation, transfection and GUS activity assayswere performed as described previously [4,36].MicroscopyTrichomes and root hairs were analyzed and photo-graphed as described [4]. The pattern of epidermal celltypes was determined as described previously [6,7,42].RNA isolation, RT-PCR and quantitative real-time PCRFor RT-PCR, total RNA was isolated from seedlings ortransfected protoplasts using the RNeasy Plant Mini Kit(QIAGEN, Mississauga, Ontario, Canada). cDNA was syn-thesized using 1 μg total RNA by Oligo(dT)-primedreverse transcription using OMNISCRIPT RT Kit (QIA-GEN). The primers used for cloning or examining theexpression of corresponding genes are as follows: TRY-specific primers: 5'-ATGGATAACACTGACCGTCG-3' and5'-CTAGGAAGGATAGATAG-3', CPC-specific primers: 5'-ATGTTTCGTTCAGACAAGGC-3' and 5'-TCATTTC-CTAAAAAAGTCCT-3', TCL1-specific primers: 5'-ATGGA-TAACACAAACCGTC-3' and 5'-TCATTTGTGGGAGAAATAGTC-3', ETC1-specific primers:5'-ATGAATACGCAGCGTAAGTC-3' and 5'-TCAACG-TAATTGAGATCTTCG-3', ETC2-specific primers: 5'-ATGGATAATACCAACCGTC-3' and 5'-TTACAATTTTA-GATTTTCTTG-3', ETC3-sepcific primers: 5'-ATGGATAAC-CATCGCAGGAC-3' and 5'-TCAATTTTTCATGACCCAAAAC-3', TTG1-specific prim-ers: 5'-ATGGATAATTCAGCTCCAG-3' and 5'-TCAAACTCTAAGGAGCTGC-3', GL1-specific primers: 5'-ATGAGAATAAGGAGAAG-3' and 5'-CTAAAGGCAG-TACTCAACATC-3', WER- specific primers: 5'-ATGA-GAAAGAAAGTAAGTAG-3' and 5'-TCAAAAACAGTGTCCATC-3', GL3-specific primers: 5'-ATGGCTACCGGACAAAACAG-3' and 5'-AAGGAACG-GGAAGCAAACCACTGTG-3', EGL3 specific primers: 5'-ATGGCAACCGGAGAAAACAGAACG-3' and 5'-TCT-CAAGGACTCCTCCAAGAAACG-3', ACTIN2 specificprimers: 5'-CCAGAAGGATGCATATGTTGGTGA-3'and 5'-GAGGAGCCTCGGTAAGAAGA-3'. The quantitative real-time PCR was performed using the MJ MiniOpticon real-time PCR system (Bio-Rad, http://www.biorad.com) andIQ SYBR Green Supermix (Bio-Rad).Authors' contributionsSW and YC isolated the double, triple, quadruple andquintuple mutants. SW and LH performed trichome androot hair analyses. SW performed the protoplast transfec-tion assays. JG participates in making constructs and per-forming plant two-hybrid protein-protein assays. JS andJ–GC conceived and coordinated the study. All authorsparticipated in drafting and editing the manuscript, andread and approved the final manuscript.AcknowledgementsWe thank Drs. Tom Guilfoyle and Gretchen Hagen (University of Missouri-Page 11 of 13(page number not for citation purposes)Columbia) for providing vectors for protoplast transfection assays, and Carol Tsang, Sophia Zhao, Vanessa Lee and Hyun-Kyung Lee (University of British Columbia) for helping in PCR genotyping. 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