A competitive complex formation mechanism underlies trichome patterning on Arabidopsis leaves

Simona Digiuni^1,a, Swen Schellmann^1,a, Florian Geier^2,3,4,a, Bettina Greese^2,3,4,a, Martina Pesch¹, Katja Wester¹, Burcu Dartan¹, Valerie Mach¹, Bhylahalli Purushottam Srinivas¹, Jens Timmer^2,5, Christian Fleck^2,4 & Martin Hulskamp¹

^aThese authors contributed equally to this work

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Synopsis

Arabidopsis trichomes are leaf hairs formed by single epidermal cells and their patterning serves as a model system for de novo pattern formation in plants. Trichomes are initiated in a regular spacing pattern in a growing field of rapidly dividing cells at the base of the young leaf. All current data indicate that the relative position of newly emerging trichomes is governed by cellular interactions between initially equivalent cells. Genetic and molecular experiments have identified genes either by promoting or inhibiting trichome fate. The trichome-promoting genes encode the R2R3 MYB transcription factors GLABRA1 (GL1) and MYB23 (Oppenheimer et al, 1991; Kirik et al, 2001, 2005), the bHLH factors GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) (Payne et al, 2000; Zhang et al, 2003; Bernhardt et al, 2005) and the WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) (Galway et al, 1994; Walker et al, 1999a). The trichome inhibitors are represented by a class of six single-repeat MYB-related transcription factors (Wada et al, 1997a; Schellmann et al, 2002; Kirik et al, 2004b; Wang et al, 2007; Tominaga et al, 2008), most importantly TRIPTYCHON (TRY) (Schellmann et al, 2002) and CAPRICE (CPC) (Wada et al, 1997a). Both groups of genes are initially expressed in all cells. It is thought that differences are generated by an activator–inhibitor-like mechanism such that the activators form a R2R3 MYB/bHLH/WD40 trimeric complex, which is counteracted by the mobile inhibitors through competition with the R2R3 MYB for binding to bHLH proteins. By integrating the key genetic and molecular data of the trichome patterning system, we develop a new theoretical model that allows the direct testing of the effect of experimental interventions and in the prediction of patterning phenotypes. We test several key assumptions of our model experimentally. We show experimentally that the trichome inhibitor TRIPTYCHON is transcriptionally activated by the known positive regulators GLABRA1 and GLABRA3. By particle bombardment of protein fusions with GFP, we show that TRIPTYCHON and CAPRICE but not GLABRA1 and GLABRA3 can move between cells. Further, we confirm the yeast two-hybrid interaction between TRY and bHLH by bimolecular fluorescence complementation assays and pull-down experiments. With both techniques, we discover a new interaction between TRY and GL1. To address the potential of this interaction, we formulate three variants of our model (cf. Figure 1) and challenge them experimentally. In the single competitive inhibition model, TRY binds to free GL3 and prevents GL1 GL3 dimerization. In the double competitive inhibition model, TRY binds to free GL3 or GL1, thereby preventing the interaction between GL1 and GL3. In the uncompetitive inhibition model, TRY binds to the GL1 GL3 dimer and represses its function. All models show similar patterning, yet for different parameter ranges and sensitivities. To determine the biological relevance of the three cases, we perform overexpression experiments. We use the 35S promoter to express TRY and GL3 in all cells and the GL2 promoter to express them trichome specific. All GL2:TRY plants are completely glabrous in two independent lines. 35S:GL3 lines show a higher trichome density in the patterning zone and GL2:GL3 lines exhibit a normal trichome density (cf. Figure 2C and D). We simulate the corresponding overexpression of TRY and GL3 using the three inhibition models (cf. Figure 2E–G). Because the parameters of these models are unknown, we randomly sample the parameter space. By exploiting our experimental results, we formulate a specific set of criteria and test each sample whether these criteria are matched. On the basis of our simulations, we conclude that the uncompetitive scenario is not consistent with our data. Moreover, we find that the single competitive inhibition is sufficient to explain all experimental findings. 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.

Figure 1

Mathematical modelling. (A) Activation part of the trichome patterning model. Solid lines indicate processes that are contained in the final model, whereas dashed lines indicate hypotheses that are rejected during the analysis. Greek letters denote the corresponding rate constants. The active complex (AC) induces the expression of the patterning genes GLABRA1 (GL1), GLABRA2 (GL2), GLABRA3 (GL3) and TRIPTYCHON (TRY). GL1 and GL3 form the active complex by dimerization. GL1, GL3 and TRY are basally expressed, and GL3 and TRY are non-cell autonomous. Basal and AC-regulated expression (green and blue arrows) denote processes that are manipulated in the simulations and experiments. (B) Inhibition part of the trichome patterning model. The three inhibition scenarios characterize how TRY may inhibit the positive feedback described in (A). In the cases of single competitive inhibition, TRY prevents the formation of the active complex by binding to free GL3, whereas in the double competitive inhibition TRY binds additionally to free GL1. In case of uncompetitive inhibition, TRY directly binds to the existing active complex. In all scenarios, the resulting inactive complex is denoted by IC. The full model comprises the interactions shown in (A) and one of the inhibitions given in (B).

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Figure 2

Expression of TRY:GUS in wild type and mutants. TRY:GUS expression is shown in young leaves: (A) wild type; (B) gl3 and (C) gl1-1. Note that the ubiquitous expression at the leaf base is absent in all single mutants.

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