Table 1.
Comparison of Trichome Patterning Phenotypes
Figure 1.
Expression and Localization Analysis of TTG1 in Developing Leaves
(A) pTTG1:GUS expression in young leaf. Inset depicts a high magnification of an area with two trichomes. Note that the expression strength is similar in all cells, including trichomes.
(B) pTTG1:GUS expression in mature leaf. Expression has ceased.
(C) pTTG1:TTG1–YFP fluorescence in young leaf. Note that in older trichomes fluorescence is still found but that epidermal cells around them have no fluorescence.
(D) Three-dimensional illustration of signal strength in (C). The fluorescence intensity is indicated by the size of the peaks.
(E) pTTG1:TTG1–YFP. The arrow depicts an incipient trichome.
(F) Quantification of the relative fluorescence intensity along the green line in (E). Note that the intensity drops the most in the cell next to the trichome.
(G) pTTG1:GFP-NLS. The arrow depicts an incipient trichome.
(H) Quantification of the relative fluorescence intensity along the pink line in (G).
(I) gl3 pTTG1:YFP. The arrow depicts an incipient trichome.
(J) Quantification of the relative fluorescence intensity along the green line in (I).
Yellow, YFP-specific fluorescence; blue, cell wall stained with propidium iodide (false colored).
Figure 2.
Phenotypic Description of Mutants and Transgenic Lines and Localization of TTG–YFP Fusion Protein
(A–D) Seed coat mucilage stained with ruthenium red. This staining visualizes the mucilage coat as a diffusely stained zone around the seed indicated by a curly bracket. (A) wild-type ecotype RLD. (B) ttg1–13, no mucilage is seen. (C) ttg1–13 pTTG1:TTG1. (D) ttg1–13 pTTG1:TTG-YFP.
(E) Seed coat color. Upper row: ttg1–13 pTTG1:TTG1, ttg1–13 pTTG1:TTG1-YFP line #4, ttg1–13 pTTG1:TTG1–YFP line #1. Lower row from left to right: wild-type ecotype RLD, strong allele ttg1–13, weak allele ttg1–9 and weak allele ttg1–10.
(F) pTTG1:TTG1–YFP fluorescence. Strong fluorescence is found in the nucleus, and moderate fluorescence in the cytoplasm. This is particularly good to see in regions containing undifferentiated cells.
(G) Western blot analysis to test the integrity of the TTG1–YFP and GFP–NLS fusion proteins. The TTG1–YFP fusion protein (70.5 kDa) is detected as a single band at the expected size (upper arrowhead). This band is not seen in the control lane ttg1–13, and no degradation products were found. Also the GFP–NLS fusion (31 kDa) is detected at the expected size (lower arrowhead).
Figure 3.
(A, C, E) Control plants after 24 h of DMSO (2%) treatment.
(B, D ,F) Plants after 24 h of epoxomicin (20 μM) treatment. (A and B) Rosette leaf (R) and basis of a cotyledon. Note the strong yellow fluorescence in the rosette leaf and the absence of yellow fluorescence in the cotyledon cells in the control (A) and nuclear fluorescence after epoxomicin treatment (B). (C and D) High magnification of cotyledon cells. (E and F) Depletion of TTG–YFP is seen in the control (E) as well as in epoxomicin treated plants (F).
(G) Quantification of the protein distribution of control plants and epoxomicin-treated plants (n = 36).
Figure 4.
The Poethig collection GAL4/VP16 activator line #232 containing a pUAS:ER-GFP and a pUAS:TTG1-YFP construct was used to test whether TTG1 moves into trichomes.
(A) GFP-specific fluorescence channel showing the expression pattern of the GAL4/VP16 driver line. Note that cells immediately next to a trichome (arrow) show strong expression (green).
(B) YFP-specific fluorescence channel showing the distribution of TTG1–YFP. Note that the trichome nuclei show strong staining.
(C) Overlay of (A) and (B) with the GFP shown in false color (red).
(D) Outline of the Cre-Lox strategy to generate mutant ttg1 sectors. TTG1 and GUS under the control of the CaMV 35S promoter were cloned between the lox sites, and ttg1 mutant plants were transformed. The ttg1 phenotype was completely rescued, and plants showed ubiquitous GUS staining (unpublished data). After saturating heat treatment, the recombination results in the deletion of the 35S:TTG1 and 35S:GUS. All daughter cells were hence ttg1 mutant and GUS-negative (unpublished data).
(E) Cre-Lox-induced sectors. Blue regions are wild-type TTG1 and white sectors are genetically ttg1 mutant. Note that trichomes are also found in white sectors.
(F) Higher magnification of (E) with trichomes in a white sector indicated by an arrow.
Figure 5.
Confocal Images of Nicotiana benthamiana Mesophyll Cells Microinjected with Fluorescent Probes
(A) Symplasmic connectivity is probed with the nucleic acid tracer acridine orange (red, RNA; green, DNA). After 1 min, DNA/RNA fluorescence staining is observed in nuclei of injected and neighboring cells.
(B) An 11-kDa rhodamine–dextran probe remains in the injected cell (red). Image was taken 10 min after injection.
(C) Recombinant TTG facilitates 11-kDa FITC–dextran (green) movement into neighboring cells. The fluorescent signal is detected in adjacent cells (*) after 1 min.
(D) After 5 min the green fluorescent tracer moves into 3–5 cells distant from the injected cell. The blue channel shows autofluorescence of plastids (false colored).
(E and F) TTG1 labeled with rhodamine (red) moves from cell to cell.
(F) Merged image showing nucleic acid (nucleus) and cell wall staining with DAPI (blue).
(G) GST labeled with rhodamine (red) remains in the injected cell and does not allow transport of 11-kDa FITC dextran (green).
(H) Merged image with DAPI staining (blue) and autofluorescence of chloroplast (green, false colored). Arrows indicate side of injection.
Table 2.
TTG1 Moves from Cell to Cell in Microinjection Assays
Figure 6.
TTG1 Movement between Cell Layers
(A) ppcA1:GFP–YFP in a young leaf. GFP/YFP-specific fluorescence is found in the subepidermal but not in the epidermal cell layer.
(B) ppcA1:GFP–YFP in an old leaf. Fluorescence is restricted to the subepidermal cell layers.
(C) ppcA1:YFP in a young leaf. YFP is found in all cells.
(D) ppcA1:YFP in an old leaf. Subepidermal expressed YFP is occasionally found in the epidermis (arrow). Trichomes show little or no fluorescence as shown in this picture.
(E) ppcA1:TTG1–YFP in a young leaf. Fluorescence is found in all cell layers.
(F) ppcA1:TTG1–YFP in an older leaf. In the epidermis, only trichomes exhibit fluorescence. Inset shows epidermis at higher magnification. Green, specific YFP fluorescence; red, chlorophyll fluorescence.
Figure 7.
Mathematical Modeling of Trichome Patterning by Depletion of TTG1
(A) Interaction scheme. TTG1 is ubiquitously expressed at rate α1 (magenta arrow), degraded at rate λ1 (red arrow), and nondirectionally transported between cells at rate d (green arrow). It forms an AC with GL3 at rate β (blue arrows). The AC induces the expression of GL3 at rate α2 (brown arrow). GL3 and AC are degraded at rates λ2 and λ3, respectively.
(B) Typical concentration pattern of total TTG1 (i.e., TTG1 + AC). Model parameters were estimated as explained in the Materials and Methods section. Light color indicates high concentration. Levels are normalized by the maximal concentration found in trichomes, which are indicated by white. A substantial amount of TTG1 is found in trichomes while it is depleted in neighboring cells.
(C) Dependence of trichome density on parameters related to TTG1 function (color code as in (A); α1, star; α2, plus; β, diamond; λ1, triangle; λ3, circle; d, square). Parameters are changed in a range from 10% to 1000% of their estimated values. Blunt ends denote the loss of trichome patterning. Inset: Corresponding change of the percentage of trichome found in clusters.
Table 3.
Parameters of the Mathematical Model