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First published online 31 March 2009
doi: 10.1242/dev.021733

Development 136, 1487-1496 (2009)
Published by The Company of Biologists 2009
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Functional diversity of R3 single-repeat genes in trichome development

Katja Wester1, Simona Digiuni1, Florian Geier2, Jens Timmer3, Christian Fleck3 and Martin Hülskamp1,*

1 University of Cologne, Botanical Institute III, Gyrhofstr. 15, 50931 Cologne, Germany.
2 University of Freiburg, Department of Biology, Schaenzlestr. 1, 79104 Freiburg, Germany.
3 University of Freiburg, Department of Mathematics and Physics, Hermann-Herder-Str. 3a, 79104 Freiburg, Germany.


Figure 1
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  		Fig. 1. Molecular and cytological expression analysis of Arabidopsis ETC3. (A) The ETC3 gene. Exons, black; introns, white. The T-DNA insertion of the salk line 094027 is located in the second intron (arrowhead). (B) RT-PCR of etc3 and wild type (WT) using primers that amplify full-length ETC3 cDNA or only the first two exons. Actin serves as a control. (C) RT-PCR of different tissues in WT and in cpc try plants to analyse the transcription level of ETC3 (30 cycles). Root material from etc3 was included to demonstrate the specificity of the primers. (D) GUS reporter expression from the ETC3 promoter in rosette leaves. The basal expression is indicated with a black arrow. The orange arrow indicates trichome-specific expression. (E) ETC3 promoter-driven GUS reporter expression in the stomata of older leaves.

 

Figure 2
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  		Fig. 2. Phenotypic analysis of trichome formation on rosette leaves in etc3 single and multiple mutants. (A) Wild-type (Col) Arabidopsis. (B) etc3. (C) p35S:ETC3 in a wild-type background. (D) cpc try. (E) etc1 etc2 etc3. (F) cpc try etc3. (G) Hypocotyl of cpc try. (H) Hypocotyl of cpc try etc3. (I) cpc try etc1 etc2. (J) cpc try etc1 etc3. (K) Phenotype of cpc try etc3 mutant plants rescued by pTRY:ETC3. (L) Phenotype of cpc try etc3 mutant plants rescued by pCPC:ETC3.

 

Figure 3
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  		Fig. 3. Localisation and movement of ETC3 protein. (A-D) Confocal laser-scanning microscopy (CLSM) of sections of the basal part of young rosette leaves in wild-type (Col) Arabidopsis plants. Cell walls in B and C are labeled in red by propidium iodide and the localisation of YFP-tagged ETC3 is in green. (A) p35S:YFP-ETC3. (B) pETC3:YFP-ETC3. Note that the trichomes and immediate neighbours show fluorescence. (C) pETC3:GFP-ER. Note that the cell-autonomous GFP-ER is restricted to trichome initials and is not found in the neighbours at the same developmental stages. (D) Transient expression of p35S:GFP-ETC3 by particle bombardment. p35S:YFP-PTS (yellow) marks the transformed cell. (E) Subepidermal expression of pRbc:YFP-ETC3 in rosette leaf (CLSM). The left-hand picture (L2) shows a subepidermal section. Chloroplasts are red owing to auto-fluorescence. The right-hand picture (L1) shows an epidermal section. (F) Trichome suppression in a wild-type background mediated by subepidermal expression of pRbc:YFP-ETC3.

 

Figure 4
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  		Fig. 4. Protein-protein interaction of ETC3 and its homologues with GL3. (A) Yeast two-hybrid assays with AD-GL3 and BD-ETC1 (1), AD-GL3 and BD-ETC2 (2), AD-GL3 and BD-ETC3 (3), AD-GL3 and BD-CPC (4), AD-GL3 and BD-TRY (5), AD-GL3 and BD-GL1{Delta} (6), AD-GL3 and pAS vector without fused protein (7), pAct vector without fused protein and pAS vector without fused protein (8), AD-SnF4 and BD-SnF1 (9). Growth indicates interaction between the respective proteins. (B) BiFC in protoplasts of GL3 with ETC1, ETC2, ETC3, CPC or TRY. Upper row, fluorescence micrographs; the lower row is an overlay of the fluorescence micrograph and the corresponding light micrograph. (C-F) Yeast three-hybrid assays. The competition between GL1 and ETC1 (1*), GL1 and ETC2 (2*), GL1 and ETC3 (3*), GL1 and CPC (4*), GL1 and TRY (5*) for binding to GL3 (fused to the GAL4 DNA-AD) is shown. The methionine-repressible promoter in the pBridge vector controls the expression of one of the CPC/TRY-like genes in the presence of GL1 (fused to GAL4 DNA-BD). (6*) The two-hybrid interaction between GL3 and GL1 in the absence of ETC3 or its homologues. (C) The interaction plate contains 0 µM methionine. No growth indicates a competition between GL1 and the respective protein. (D-F) The interaction plate contains 100 (D), 250 (E) or 500 (F) µM methionine. The methionine-sensitive promoter is inactive at 500 µM methionine and growth indicates an interaction between GL3 and GL1.

 

Figure 5
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  		Fig. 5. Modelling of the effect of protein depletion on the mobility of inhibitors. Decay of inhibitor concentration with distance from the source for inhibitors with different binding affinities for GL3. The movement ability of the inhibitor is measured by the characteristic decay length (CDL), {alpha}, which is the distance from the source at which the protein level drops to 1/e (37%) of its source level. All length scales are relative to {alpha}0, which is the CDL in the absence of protein binding (dashed line, black curve). Colour coding: absence of protein binding (black), relative binding affinity {gamma}=0.1 (red), {gamma}=1 (green), {gamma}=10 (blue), {gamma}=100 (magenta). The inset shows the dependence of the CDL on the relative binding affinity, {gamma}. Note the correspondingly coloured circles in the inset and the main graph. As the relative binding affinity of the inhibitor for the activator increases, its CDL decreases (see Materials and methods).

 

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