13 research outputs found
Trichome patterning in Arabidopsis thaliana: Mechanism and the role of TTG1 depletion/trapping
Trichome patterning in Arabidopsis thaliana is a potential model system to study
two dimensional patterning. Theoretically, lateral inhibition during trichome
patterning can be achieved either by active inhibition or by removal of trichome
promoting activity (e.g. depletion). Recent data have suggested a role of
this activator depletion mechanism in trichome patterning. It was shown that
the TTG1 protein is depleted in the trichome surrounding epidermal cells and
accumulates in the trichome initials. In this study I focused on the characterization
of the molecular mechanism and the role of TTG1 trapping during trichome
patterning.
I showed that the removal of the bHLH factor GL3 results in the abolition of the
TTG1 depletion strongly suggesting that TTG1 depletion is GL3 dependent. Cells
expressing high levels of GL3 show a strong positive effect on nuclear localization
of the TTG1 protein. GL3 also counteracts the TTG1 mobility both within as well
as between the tissue layers. Co-expression of GL3 and TTG1 in the subepidermis
blocked the mobility of TTG1 from the subepidermis to the epidermis. Within the
epidermis the TTG1 protein in the trichome initials is less free to move compared
to TTG1 in the other epidermal cells. Similarly the TTG1 entering into the
trichome initial is retained more efficiently than the TTG1 entering into other
epidermal cells. This correlates with the expression and localization pattern of
GL3 which is predominantly expressed in trichome initials. This observation was
further strengthened by p35S::GL3 lines where the depletion was lost because
of the trapping of TTG1 in all epidermal cells. Taken together these data are
clearly pointing towards a GL3 mediated nuclear trapping of TTG1 in the trichome
initials.
Weak alleles of ttg1, which produce trichome clusters, were used to test the
biological relevance of the depletion. Interestingly the weak allelic forms of TTG1
showed either a weak or no interaction with GL3 and failed to be trapped in the
nucleus in the yeast system. This led to the postulation that in these weak alleles
the TTG1 interaction with GL3 might be sufficient enough to initiate trichomes but not strong enough to attract/trap TTG1 in the nucleus resulting in no depletion of
the activator TTG1 in the trichome adjacent cells and thereby leading to cluster
formation.
A threshold level of TTG1 concentration in the nucleus appears to be crucial for the
correct branching of the trichome. This assumption also correlates nicely with the
underbranched phenotype in the weak ttg1 alleles where also nuclear TTG1 would
be expected to be less because of weak/no interaction with GL3.
The mobility domain in TTG1, which is not solely but partially responsible for the
TTG1 mobility between the tissue layers was mapped to few amino acids in the
N-terminus of TTG1.
A potential TTG1 transport inhibitor (tti) mutant was isolated in the EMS
mutagenesis screening of the ttg1pRBC::TTG1 plants that specifically inhibited the
transport of TTG1 from the subepidermis to the epidermis in leaf and the seeds.
The application of the photoconvertible marker KikGR1 in plants was shown for
the first time. This was then successfully used to study the mobility of TTG1 in the
leaf epidermis
Two-Dimensional Patterning by a Trapping/Depletion Mechanism: The Role of TTG1 and GL3 in Arabidopsis Trichome Formation
Trichome patterning in Arabidopsis serves as a model system to study how single cells are selected within a field of initially equivalent cells. Current models explain this pattern by an activator–inhibitor feedback loop. Here, we report that also a newly discovered mechanism is involved by which patterning is governed by the removal of the trichome-promoting factor TRANSPARENT TESTA GLABRA1 (TTG1) from non-trichome cells. We demonstrate by clonal analysis and misexpression studies that Arabidopsis TTG1 can act non-cell-autonomously and by microinjection experiments that TTG1 protein moves between cells. While TTG1 is expressed ubiquitously, TTG1–YFP protein accumulates in trichomes and is depleted in the surrounding cells. TTG1–YFP depletion depends on GLABRA3 (GL3), suggesting that the depletion is governed by a trapping mechanism. To study the potential of the observed trapping/depletion mechanism, we formulated a mathematical model enabling us to evaluate the relevance of each parameter and to identify parameters explaining the paradoxical genetic finding that strong ttg1 alleles are glabrous, while weak alleles exhibit trichome clusters
Nuclear trapping by GL3 controls intercellular transport and redistribution of TTG1 protein in Arabidopsis
Trichome patterning on Arabidopsis leaves is one of the best-studied model systems for two-dimensional de novo patterning. In addition to an activator-inhibitor-related mechanism, we previously proposed a depletion mechanism to operate during this process such that GLABRA3 (GL3) traps the trichome-promoting factor TRANSPARENT TESTA GLABRA1 (TTG1) in trichomes that, in turn, results in a depletion of TTG1 in trichome neighbouring cells. In this manuscript we analyze the molecular basis underlying this trapping mechanism. We demonstrate the ability of GL3 to regulate TTG1 mobility by expressing TTG1 and GL3 in different tissue layers in different combinations. We further show that TTG1 trapping by GL3 is based on direct interaction between both proteins and recruitment in the nucleus
Stochastic gene expression in Arabidopsis thaliana
Although plant development is highly reproducible, some stochasticity exists. This developmental stochasticity may be caused by noisy gene expression. Here we analyze the fluctuation of protein expression in Arabidopsis thaliana. Using the photoconvertible KikGR marker, we show that the protein expressions of individual cells fluctuate over time. A dual reporter system was used to study extrinsic and intrinsic noise of marker gene expression. We report that extrinsic noise is higher than intrinsic noise and that extrinsic noise in stomata is clearly lower in comparison to several other tissues/cell types. Finally, we show that cells are coupled with respect to stochastic protein expression in young leaves, hypocotyls and roots but not in mature leaves. Our data indicate that stochasticity of gene expression can vary between tissues/cell types and that it can be coupled in a non-cell-autonomous manner
Identification of the Trichome Patterning Core Network Using Data from Weak ttg1 Alleles to Constrain the Model Space
The regular distribution of trichomes on leaves in Arabidopsis is a well-understood model system for two-dimensional pattern formation. It involves more than 10 genes and is governed by two patterning principles, the activator-inhibitor (AI) and the activator-depletion (AD) mechanisms, though their relative contributions are unknown. The complexity of gene interactions, protein interactions, and intra- and intercellular mobility of proteins makes it very challenging to understand which aspects are relevant for pattern formation. In this study, we use global mathematical methods combined with a constraining of data to identify the structure of the underlying network. To constrain the model, we perform a genetic, cell biological, and biochemical study of weak ttg1 alleles. We find that the core of trichome patterning is a combination of AI and AD mechanisms differentiating between two pathways activating the long-range inhibitor CPC and the short-range inhibitor TRY
Confocal Images of Nicotiana benthamiana Mesophyll Cells Microinjected with Fluorescent Probes
<div><p>(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.</p>
<p>(B) An 11-kDa rhodamine–dextran probe remains in the injected cell (red). Image was taken 10 min after injection.</p>
<p><b>(</b>C) Recombinant TTG facilitates 11-kDa FITC–dextran (green) movement into neighboring cells. The fluorescent signal is detected in adjacent cells (*) after 1 min.</p>
<p>(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).</p>
<p>(E and F) TTG1 labeled with rhodamine (red) moves from cell to cell.</p>
<p>(F) Merged image showing nucleic acid (nucleus) and cell wall staining with DAPI (blue).</p>
<p>(G) GST labeled with rhodamine (red) remains in the injected cell and does not allow transport of 11-kDa FITC dextran (green).</p>
<p>(H) Merged image with DAPI staining (blue) and autofluorescence of chloroplast (green, false colored). Arrows indicate side of injection.</p></div
TTG1 Movement between Cell Layers
<div><p>(A) ppcA1:GFP–YFP in a young leaf. GFP/YFP-specific fluorescence is found in the subepidermal but not in the epidermal cell layer.</p>
<p>(B) ppcA1:GFP–YFP in an old leaf. Fluorescence is restricted to the subepidermal cell layers.</p>
<p>(C) ppcA1:YFP in a young leaf. YFP is found in all cells.</p>
<p>(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.</p>
<p>(E) ppcA1:TTG1–YFP in a young leaf. Fluorescence is found in all cell layers.</p>
<p>(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.</p></div
TTG1 Movement
<div><p>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.</p>
<p>(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).</p>
<p>(B) YFP-specific fluorescence channel showing the distribution of TTG1–YFP. Note that the trichome nuclei show strong staining.</p>
<p>(C) Overlay of (A) and (B) with the GFP shown in false color (red).</p>
<p>(D) Outline of the Cre-Lox strategy to generate mutant <i>ttg1</i> sectors. <i>TTG1</i> and <i>GUS</i> under the control of the CaMV <i>35S</i> promoter were cloned between the lox sites, and <i>ttg1</i> mutant plants were transformed. The <i>ttg1</i> phenotype was completely rescued, and plants showed ubiquitous GUS staining (unpublished data). After saturating heat treatment, the recombination results in the deletion of the <i>35S:TTG1</i> and <i>35S:GUS</i>. All daughter cells were hence <i>ttg1</i> mutant and GUS-negative (unpublished data).</p>
<p>(E) Cre-Lox-induced sectors. Blue regions are wild-type <i>TTG1</i> and white sectors are genetically <i>ttg1</i> mutant. Note that trichomes are also found in white sectors.</p>
<p>(F) Higher magnification of (E) with trichomes in a white sector indicated by an arrow.</p></div