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.

Trichome patterning in Arabidopsis serves as a model system for de novo pattern formation in plants.It is thought to typify the theoretical activator-inhibitor mechanism, although this hypothesis has never been challenged by a combined experimental and theoretical approach. By integrating the key genetic and molecular data of the trichome patterning system, we developed a new theoretical model that allows the direct testing of the effect of experimental interventions and in the prediction of patterning phenotypes. We show experimentally that the trichome inhibitor TRIPTYCHON is transcriptionally activated by the known positive regulators GLABRA1 and GLABRA3. Further, we demonstrate by particle bombardment of protein fusions with GFP that TRIPTYCHON and CAPRICE but not GLABRA1 and GLABRA3 can move between cells. Finally, theoretical considerations suggest promoter swapping and basal overexpression experiments by means of which we are able to discriminate three biologically meaningful variants of the trichome patterning model. Our study demonstrates that the mutual interplay between theory and experiment can reveal a new level of understanding of how biochemical mechanisms can drive biological patterning processes.

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ORCID IDs: 0000-0002-6592-4445 (J.F.H.); 0000-0001-6633-9769 (M.K.); 0000-0002-1317-7716 (R.S.); 0000-0001-7734-3771 (S.W.-P.).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.One well-studied example for a single regulatory protein complex driving the evolution of multiple traits in plants is the R2R3MYB/basic helix-loop-helix (bHLH)/WD40 (MBW) complex (Broun, 2005;Koes et al., 2005;Ramsay and Glover, 2005;Serna and Martin, 2006;Feller et al., 2011). Together, the corresponding three genes are required for the regulation of metabolic pathways (anthocyanin and proanthocyanidin production) and the differentiation of epidermal cell types in higher plants (Broun, 2005;Koes et al., 2005;Ramsay and Glover, 2005;Serna and Martin, 2006;

Protein-protein interactions are fundamental to virtually every aspect of cellular functions. Blue, green and yellow bimolecular fluorescence complementation (BiFC) systems based on GFP and its variants allow the investigation of protein-protein interactions in vivo. We have developed the first red BiFC system based on an improved monomeric red fluorescent protein (mRFP1-Q66T), expanding the range of possible applications for BiFC.With interactome data available for several model organisms, a challenging next step in post-genomic research is to analyze protein complex formation in vivo. The recently developed BiFC assay is a comparably fast and simple noninvasive technology to study protein interactions inside living cells. BiFC is based on the reconstitution of the yellow fluorescent protein (YFP) from two nonfluorescent fragments when they are brought into close proximity by a physical interaction between proteins fused to each fragment 1 . This approach has proven to be robust and versatile, and multicolor versions using spectral variants of GFP further increase the potential of this technology 2 . The color spectrum available for BiFC, however, has been limited to blue, green and yellow.The red fluorescent protein from Discosoma sp. (DsRED) and its variants are established intracellular reporter proteins, but the characteristics of most DsRED variants, particularly their obligate tetramerization, impede their application in BiFC 3,4 . An extensively mutated monomeric DsRED variant (mRFP1) has been generated 5 , but substantially altered spectra, poor brightness and low photostability limit its usefulness as a reporter protein.We have identified improved mRFP1 mutants, which allowed us to establish a red fluorescent reporter system for detection of protein interactions in vivo. We used site-directed mutagenesis to generate mRFP1 variants with a randomized first position of the fluorophore. Screening of 5,000 colonies resulted in the identification of 50 clones exhibiting strong red fluorescence representing three different mutations (Q66C, Q66S and Q66T) with frequencies of 5%, 43% and 52%, respectively.