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First published online October 12, 2007
doi: 10.1242/10.1242/dev.009597

Development 134, 3873-3882 (2007)
Published by The Company of Biologists 2007
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TRICHOMELESS1 regulates trichome patterning by suppressing GLABRA1 in Arabidopsis

Shucai Wang1, Su-Hwan Kwak2, Qingning Zeng1, Brian E. Ellis3, Xiao-Ya Chen4, John Schiefelbein2,* and Jin-Gui Chen1,*

1 Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada.
2 Department of Molecular, Cell, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
3 Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4, Canada.
4 National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.


Figure 1
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  		Fig. 1. tcl1-1D is a gain-of-function dominant glabrous mutant. (A) Trichomes on leaves of WS (top) and gpa1-2 mutant (middle), and glabrous leaves of tcl1-1D/gpa1-2 dominant mutant (bottom). (B) Trichomes on leaves of Col (top) and glabrous leaves of transgenic Arabidopsis plants overexpressing HA-TCL1 (bottom). The overexpression of TCL1 was confirmed by RT-PCR (below) using a pair of primers - one HA-specific, the other TCL1-specific. Expression of ACTIN2 provided a control. (C) Root hair production in WS, gpa1-2, Col, in the tcl1-1D/gpa1-2 dominant mutant and in a transgenic plant overexpressing HA-TCL1.

 

Figure 2
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  		Fig. 2. Molecular cloning of TCL1. (A) Diagram illustrating the insertion site of the activation-tagged T-DNA in the tcl1-1D/gpa1-2 mutant. The orientation of the four 35S-enhancer repeats in the T-DNA situated 2115 bp upstream of the start codon of TCL1 (At2g30432) is indicated by arrows. (B) RT-PCR indicating transcript level of TCL1 in wild-type, gpa1-2 and tcl1-1D/gpa1-2 Arabidopsis plants. RNA was isolated from 10-day-old light-grown seedlings. ACTIN2 provided a control. (C) The TCL1 protein is similar to other single-repeat R3 MYB transcription factors TRY, CPC, ETC1, ETC2 and At4g01060. Identical amino acids are shaded in black, similar amino acids in gray. The amino acid signature [D/E]Lx2[R/K]x3Lx6Lx3R (Zimmermann et al., 2004Go) that is required for interacting with R/B-like bHLH transcription factors is indicated by arrowheads. Asterisks indicate the amino acids within the MYB domain that are crucial for cell-to-cell movement (Kurata et al., 2005Go). (D) Phylogenetic analysis of TCL1, TRY, CPC, ETC1, ETC2 and At4g01060.

 

Figure 3
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  		Fig. 3. Loss-of-function allele of TCL1. (A) Diagram illustrating the T-DNA insertion site in the tcl1-1 mutant. The T-DNA is inserted in the second intron of TCL1. (B) Expression of TCL1 in wild-type and tcl1-1 mutant Arabidopsis plants. Expression of ACTIN2 provided a control. (C) Comparison of trichome formation on the first pedicel and the internode subtending the site of the first flower on the main inflorescence of wild type (left), tcl1-1 mutant (middle) and tcl1-1 mutant transformed with PTCL1:TCL1-GFP (right). Arrowheads indicate the site of the first flower/siliques on the main inflorescence stem. Arrows indicate trichomes formed on the main inflorescence stem and pedicel of the tcl1-1 mutant. Beneath is shown the results from RT-PCR in which GFP-specific primers were used to examine the expression of TCL1-GFP in PTCL1:TCL1-GFP/tcl1-1 plants. Expression of ACTIN2 provided a control. (D) TCL1-GFP fluorescence in the epidermal cells of the lower part of the hypocotyl in a 4-day-old PTCL1:TCL1-GFP seedling. Shown are differential interference contrast (left) and confocal (right) images of hypocotyl epidermal cells. Arrows point to the nucleus of a cell.

 

Figure 4
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  		Fig. 4. Trichomes on the main inflorescence stem and pedicels of tcl1-1 cpc double mutants. (A) The regions that were used to score trichome density. Arrowhead indicates the site of the first flower/silique on the main inflorescence stem. The positions before (0) and after (first to fourth) the site of the first flower/silique on the main inflorescence stem are indicated. These five positions were used to score the number of trichomes on the main inflorescence stem. The first seven siliques (numbered 1 to 7) on the main inflorescence stem were used to examine the number of trichomes on pedicels. (B) Trichome density on the internode before (0) and after (first to fourth) the site of the first flower on the main inflorescence stem of wild type and mutants. Shown are means±s.e. of at least nine plants. *, significantly different from tcl1-1 mutants, P<0.05. (C) Trichome density on pedicels on the main inflorescence. No trichome was found on pedicels in wild type, try, or cpc single mutants. Shown are means±s.e. of at least nine plants. *, significantly different from tcl1-1 mutants, P<0.05.

 

Figure 5
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  		Fig. 5. Trichomes on the main inflorescence stem and pedicels of tcl1-1 cpc try triple mutants. Upper panels show the top portions of the main inflorescence stem of wild-type (Col), tcl1-1, tcl1-1 cpc, and tcl1-1 cpc try mutants. Bottom panels are magnified images of the inflorescence stem.

 

Figure 6
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  		Fig. 6. TCL1 suppresses the expression of GL1. (A) RT-PCR analysis of TTG1, GL1, GL2 and GL3 transcripts in Arabidopsis seedlings overexpressing HA-TCL1. Expression of ACTIN2 provided a control. (B) Suppression of GL1::GUS reporter by TCL1. Shown are the expression of GL1::GUS reporter in wild-type (Col) and in 35S:HA-TCL1 backgrounds. In the GL1::GUS reporter, the expression of GUS was driven by a chimerical genomic sequence consisting of the immediate 5' upstream sequence, the first intron, and the 3'-UTR region of GL1 (Wang et al., 2004Go). (C) RT-PCR analysis of TCL1 transcript in various tissues/organs. Expression of ACTIN2 provided a control. (D) RT-PCR analysis of GL1 and GL2 transcripts in the developing inflorescence of tcl1-1 mutants. Expression of ACTIN2 provided a control.

 

Figure 7
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  		Fig. 7. GL1 is a target gene of TCL1. (A) Schematic showing the effectors and reporter constructs used in the Arabidopsis protoplast transfection assays. (B) TCL1-VP16 transactivator fusion protein activates the expression of the reporter. Effectors and reporters were cotransfected into protoplasts derived from Arabidopsis rosette leaves. GUS activity was assayed after protoplasts were incubated in the dark for 20-22 hours. (C) Leaves and stem trichome phenotypes of transgenic plants overexpressing TCL1-VP16. (D) RT-PCR analysis of the level of GL1, TTG1, GL3 and GL2 transcripts in plants overexpressing TCL1-VP16. Expression of ACTIN2 provided a control. (E) Chromatin immunoprecipitation (ChIP) assay. ChIP was performed with 35S:HA-TCL1 plants using anti-HA antibodies. Rabbit preimmune serum was used as a mock control. Primer sets specific for the first intron or the 3'-UTR region of GL1 were used in PCR reactions. ACTIN2 provided a control.

 

Figure 8
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  		Fig. 8. Model of action of TCL1 in trichome patterning in Arabidopsis. GL1 encodes an R2R3 MYB-type transcription factor. GL3 encodes a bHLH-related transcription factor. TTG1 encodes a WD40 domain-containing protein. GL2 encodes a homeobox transcription factor. TCL1 encodes a single-repeat R3 MYB transcription factor. Other known single-repeat MYB transcription factors, including TRY, CPC, ETC1 and ETC2, are not shown in the model. TTG1, GL3 and GL1 form an activator complex, whereas TCL1, TRY, CPC, ETC1 and ETC2 act as repressors that were proposed to bind GL3, thus limiting the interaction between GL1 and GL3, an interaction that is required to form the TTG1-GL3-GL1 activator complex that regulates the transcription of GL2. As shown in this study, TCL1 can directly suppress the expression of GL1, thereby limiting the transcriptional activity of the TTG1-GL3-GL1 activator complex. It is unclear whether the TTG1-GL3-GL1 activator complex can promote the transcription of TCL1. Arrows indicate positive regulation and the blunt-ended line indicates negative regulation.

 




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