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First published online 23 April 2008
doi: 10.1242/dev.016873

Development 135, 1991-1999 (2008)
Published by The Company of Biologists 2008
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The TTG1-bHLH-MYB complex controls trichome cell fate and patterning through direct targeting of regulatory loci

Mingzhe Zhao1, Kengo Morohashi2, Greg Hatlestad1, Erich Grotewold2 and Alan Lloyd1,*

1 Section of Molecular Cell and Developmental Biology and The Institute for Cellular and Molecular Biology, The University of Texas at Austin, 2500 Speedway, Austin, TX 78712, USA.
2 Department of Plant Cellular and Molecular Biology and Plant Biotechnology Center, The Ohio State University, Columbus, OH 43210, USA.


Figure 1
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  		Fig. 1. Expression pattern of YFP-TTG1 fusion in the leaf epidermis. Maximum intensity projection images of confocal stacks of a TTG1::YFP-TTG1 construct in developing leaves of 20-day-old ttg1 mutant seedlings. (A) Trichome initials (arrows) and surrounding cells. (B) Overview of a developing young leaf. This leaf is not flat so that in some areas the pavement cells are in focus and in other areas, focus is higher up on the trichomes. (C) Mature trichomes and surrounding cells. Scale bars: 10 µm in A; 100 µm in B; 50 µm in C.

 

Figure 2
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  		Fig. 2. Direct activation of GL3 target genes by 35S::TTG1-GR. (A) Gene expression levels were measured by relative quantitative PCR. The results were calculated using the comparative Ct method (ABI bulletin) and presented as fold changes compared with the mock or CHX treatment, which were standardized to the level of actin expression. The induced expression levels of GL2, TTG2, CPC and ETC1 were statistically significantly different from those of control treatments (P<0.05); error bars indicate the ranges of expression change; nt, not tested. (B) Semi-quantitative PCR of ChIP experiments using ttg1/pTTG1::YFP-TTG1 (left) or gl1/pGL1::GL1-YFP-cMYC (right). PCRs were performed on three fourfold serial dilutions of the immunoprecipitated material, represented by the black slope.

 

Figure 3
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  		Fig. 3. Co-precipitation of TTG1-cMYC with HA-GL3-6His and GL1-YFP-6His from seedling extracts. His-Select Ni columns were used to pull down 6His-tagged fusion proteins. Input and eluted proteins were separated by SDS-PAGE gels in the order labeled. Membranes were probed with anti-cMYC mAb. In vivo interaction between TTG1 and GL3 is indicated in A and between TTG1 and GL1 in B.

 

Figure 4
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  		Fig. 4. Speckled nuclear distribution of GL3 in the leaf epidermis of ttg1 and gl1 mutants. Confocal images of (A) gl3/pGL3::GL3-YFP, (B) ttg1/pGL3::GL3-YFP, (C) ttg1/pGL1::GL1-YFP-cM, (D) gl1/pGL3::GL3-YFP and (E) gl1/pGL3::GL3-YFP. GL3-YFP is unevenly distributed and forms nuclear speckles in leaf epidermal cells of ttg1 and gl1 mutants (B,D). GL1-YFP formed only a couple of speckles in nuclei in occasional leaf epidermal cells (C). Uniform GL3-YFP distribution in gl3 leaf epidermal cells (A), and gl1 root epidermal cells (inset picture shows GL3::GL3-YFP accumulation in hairless cell files forming wild type-looking stripes) (D). Scale bars: 10 µm.

 

Figure 5
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  		Fig. 5. Intercellular trafficking of YFP-CPC in the leaf epidermis. Confocal images of bombarded YFP fusion proteins. (A-E) YFP fluorescence (green). (F-J) Chlorophyll fluorescence (red). (K-O) Merged images. Only YFP-CPC shows cell-to-cell movement forming a cluster of fluorescent cells (D,N). TTG1, GL3, GL1 and GL2 fusions are cell autonomous, as fluorescence is restricted to single cells. Guard cells, as in the upper left of B, are often autofluorescent under these conditions. Scale bars: 10 µm.

 

Figure 6
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  		Fig. 6. GL3 and EGL3 have overlapping but distinct expression patterns. (A-D) Transcription patterns of GL3 and EGL3 promoter::GUS in wild-type leaves. (A,C) At a very young stage, both GL3 and EGL3 are strongly transcribed in undifferentiated leaf primordia; in later differentiating leaves, high GL3 is observed in undifferentiated cells close to the basal edge of the leaf, while EGL3 is more widespread and not restricted to the edge; trichome initials show higher levels of GL3 and EGL3 than surrounding epidermal cells. (B,D) At a more mature stage, strong GL3 expression becomes restricted to trichomes, while EGL3 expression persists in pavement cells as well as in trichomes. (E-H) Protein accumulation patterns of GL3 and EGL3 in wild-type leaves. (E,F) GL3-YFP highly accumulates in trichome initials (arrowheads) and young trichomes. (G,H) At the same stages, strong EGL3-YFP was detected in the trichomes (arrowheads) and non-trichome cells. Scale bars: 20 µm in E,G; 50 µm in F,H.

 

Figure 7
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  		Fig. 7. Model for Arabidopsis trichome/non-trichome cell fate specification. Regulators of trichome fate are green shades, activators (GL2/TTG2) are yellow and inhibitors (CPC/ETC1) are orange. Black arrows indicate transcriptional activation. In trichome cells, the inhibitors are directly activated by the activating complex and move (broken lines) into neighboring cells, where they and endogenous inhibitors block the activity of the activating complex thereby decreasing the expression of GL2/TTG2 to below a required initiating threshold level (broken arrows). Thus, the trichome cell fate is not triggered.

 

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© The Company of Biologists Ltd 2008