Role of TRIPTYCHON in trichome patterning in Arabidopsis

<p>Abstract</p> <p>Background</p> <p>Trichome patterning in <it>Arabidopsis thaliana </it>is governed by three types of activators, R2R3MYB, bHLH and WD40 proteins, and six R3MYB inhibitors. Among the inhibitors <it>TRIPTYCHON </it>(<it>TRY</it>) seems to fulfill a special function. Its corresponding mutants produce trichome clusters whereas all other inhibitors are involved in trichome density regulation.</p> <p>Results</p> <p>To better understand the role of <it>TRY </it>in trichome patterning we analyzed its transcriptional regulation. A promoter analysis identified the relevant regulatory region for trichome patterning. This essential region contains a fragment required for a double negative feedback loop such that it mediates the repression of <it>TRY</it>/<it>CPC </it>auto-repression. By transforming single cells of <it>pTRY</it>:GUS lines with <it>p35S</it>:<it>GL1, p35S</it>:<it>GL3 </it>and <it>p35S</it>:<it>TTG1 </it>in the presence or absence of <it>p35S</it>:<it>TRY </it>or <it>p35S</it>:<it>CPC </it>we demonstrate that TRY and CPC can suppress the <it>TRY </it>expression without the transcriptional down regulation of the activators. We further show by promoter/CDS swapping experiments for the R3MYB inhibitors TRY and CPC that the TRY protein has specific properties relevant in the context of both, cluster formation and trichome density.</p> <p>Conclusions</p> <p>Our identification of a <it>TRY </it>promoter fragment mediating a double negative feedback loop reveals new insight in the regulatory network of the trichome patterning machinery. In addition we show that the auto-repression by TRY can occur without a transcriptional down regulation of the activators, suggesting that the differential complex formation model has a biological significance. Finally we show that the unique role of TRY among the inhibitors is a property of the TRY protein.</p

Trichom- und Wurzelhaar-Musterbildung in Arabidopsis thaliana. Vergleichende Promotoranalyse der Aktivatoren GLABRA1 und WEREWOLF, sowie der Inhibitoren TRIPTYCHON und CAPRICE

Die transkriptionelle Regulation der Gene GL1 und WER, als Aktivatoren der Trichom- und Wurzelhaarmusterbildung, sowie der Gene TRY und CPC, als Inhibitoren dieser beiden Musterbildungssysteme, wurde durch Eingrenzung der regulatorischen Sequenzen über Promotor-GUS- und Rettungsexperimente untersucht. Dabei konnte festgestellt werden, dass die Aktivatoren einer sehr komplexen Regulation unterliegen, während die beiden Inhibitoren, die homologe Gene darstellen, wesentlich einfacher reguliert werden. Im Gegensatz zu den sehr ähnlichen Expressionsmustern der Gene TRY und CPC, werden sie aber durch unterschiedliche Faktoren reguliert und auch ihre Minimalpromotoren unterscheiden sich sehr deutlich. Mutmaßliche cis-regulatorische Elemente, sowie die zugehörigen, bindenden Faktoren konnten vorhergesagt werden. Teilweise konnte durch Mutagenese dieser Elemente ihre Relevanz in vivo bestätigt werden

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

The Second Intron Is Essential for the Transcriptional Control of the Arabidopsis thaliana GLABRA3 Gene in Leaves

The GLABRA3 gene is a major regulator of trichome patterning in Arabidopsis thaliana. The regulatory regions important for the trichome-specific expression of GL3 have not been characterized yet. In this study, we used a combination of marker and rescue constructs to determine the relevant promoter regions. We demonstrate that a 1 kb 5' region combined with the second intron is sufficient to rescue the trichome mutant phenotype of gl3 egl3 mutants. Swap experiments of the second intron suggest that it is not sufficient to generally enhance the expression level of GL3. This implies that the second intron contains regulatory regions for the temporal and spatial regulation of GL3. The corresponding GUS-marker constructs revealed trichome-specific expression in young trichomes

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

TRANSPARENT TESTA GLABRA1 and GLABRA1 Compete for Binding to GLABRA3 in Arabidopsis

The MBW (for R2R3MYB, basic helix-loop-helix [bHLH], and WD40) genes comprise an evolutionarily conserved gene cassette that regulates several traits such as (pro) anthocyanin and anthocyanin biosynthesis and epidermal cell differentiation in plants. Trichome differentiation in Arabidopsis (Arabidopsis thaliana) is governed by GLABRA1 (GL1; R2R3MYB), GL3 (bHLH), and TRANSPARENT TESTA GLABRA1 (TTG1; WD40). They are thought to form a trimeric complex that acts as a transcriptional activation complex. We provide evidence that these three MBW proteins form either GL1 GL3 or GL3 TTG1 dimers. The formation of each dimer is counteracted by the respective third protein in yeast three-hybrid assays, pulldown experiments (luminescence-based mammalian interactome), and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer studies. We further show that two target promoters, TRIPTYCHON (TRY) and CAPRICE (CPC), are differentially regulated: GL1 represses the activation of the TRY promoter by GL3 and TTG1, and TTG1 suppresses the activation of the CPC promoter by GL1 and GL3. Our data suggest that the transcriptional activation by the MBW complex involves alternative complex formation and that the two dimers can differentially regulate downstream genes