A coherent feed-forward loop in the Arabidopsis root stem cell organizer regulates auxin biosynthesis and columella stem cell maintenance

a: Schematic representation of the HAN promoter and the corresponding deletion constructs. ++, inducible expression in CCs by 35S:WOX5-GR similar to the complete pHAN:3xGFP, +, weakly inducible, - not inducible. b: Outline of the central 4 CCs used for signal quantification. c-d: Results of the two experiments comparing representative lines from pΔ2HAN to pΔ6HAN (d) and from pΔ5HAN to pΔ8HAN (c). Values show the relative increase in GFP signal measured after 24 h of 35S:WOX5-GR induction. WOX5-GR represents a negative control without GFP reporter. x/y, roots analyzed without (x) and with (y) DEX induction. Letters denote statistical differences at p < 0.001 compared to the “full promoter” by one-way ANOVA and Dunnett’s posthoc test. In 1c, p_{no reporter} < 0.0001, p_Δ5#2 < 0.0001, p_Δ6 < 0.0001, p_Δ7 < 0.0001, p_Δ8 < 0.0001 In 1d, p_{no reporter} < 0.0001, p_Δ2 = 0.634, p_Δ3 = 0.174, p_Δ4 = 0.999, p_Δ5#1 = 0.989, p_Δ5#2 < 0.0001, p_Δ6#1 < 0.0001, p_Δ6#2 < 0.0001. e-f: Induction of 35S:LhGR pOp:WOX5-3xFlag; Q1630::H2B-tdTomato (35S::WOX5-GR) line causes relative enrichment of H3K9Ac deposition on the distal regulatory element (p2: 5021 bp upstream to ATG) of the HAN gene relative to Q1630::H2B-tdTomato as negative control. By contrast, the P1 site (−654) and H3K27me3 marks were unaffected. The data shown are means of three independent biological replicates. Error bars denote SD. In e, n.s., not significant; p_P1 = 0.267, p_P2 = 0.0152, p_ACT2 = 0.778 by a two-sided Student’s t-test. Scale bar: 20 μm.

a-b: The reduced expression of QC184 and the accumulation of starch grains in the subjacent cell layer of 5-day-old han-30 roots (a) are complemented by pHAN:HAN (b). Numbers denote the frequencies of the shown phenotypes in independent transformants. The QC is outlined in white. The restored CSCs are outlined in yellow (b). c: Representative confocal images showing accumulation of starch-positive amyloplasts (red arrowheads) and QC divisions (yellow arrowheads) of the indicated genotypes after mPS-PI-staining of 6-day-old roots. White arrowhead shows the CSC layer and black arrowhead shows the QC position in the wild type. d: Percentages of roots with indicated numbers of starch-free CSC-like layers in 6-day-old Col-0 wild type (n = 44), han-1 (n = 35), and han-30 (n = 42) roots. ***, p < 0.001, comparing presence vs. absence of starch-free CSC layers by a two-sided Fisher’s exact test with Bonferroni correction for multiple testing. n.s., not significant; p_{Col-0 vs han-1} = 0.0003, p_{Col-0 vs han-30} = 0.0003, p_{han-1 vs han-30} = 0.0650. e: Quantification of QC division in 6-day-old Col-0 wild-type (n = 43), han-1 (n = 34) and han-30 (n = 41) roots. n.s., not significant; p_{Col-0 vs han-1} = 0.0003, p_{Col-0 vs han-30} = 0.0075, p_{han-1 vs han-30} = 0.271 by a two-sided Fisher’s exact test with Bonferroni correction for multiple testing. Scale bars: 20 μm.

a: pWOX5»HAN expression in Col-0 QC184 after DEX induction. Confocal images showing erDsRed expression in mock-treated roots and after germination on 5 µM DEX for six days (a). Yellow arrowheads indicate QC position. b: Col-0 QC184 roots are unaffected by the induction of pWOX5»HAN. c-d: QC184 expression is not affected by germination on 5 µM DEX-containing medium for six days in the indicated genotypes. e-f: pWOX5:NLS-GUS (e) and QC25 (f) expression is not affected by germination on 5 µM DEX-containing medium. GUS signals (b-f) are shown in blue, and starch granules after Lugol staining in purple. In a-f, The images shown are representative of n > 20 independent roots that were measured and produced similar results. Scale bars: 20 μm.

a: Expression of pWOX5»HAN in three independent transformants in the wox5-1 background. b: Expression of pWOX5»WOX5 in three independent transformants in the wox5-1 background. -DEX, mock treated; +DEX, grown for six days after germination on 5 µM DEX. DsRED signals and red color was equally enhanced for better visualization. In a,b, The images shown are representative of n > 20 independent roots that were measured and produced similar results. Scale bars: 25 µm.

a-b: Representative confocal images showing accumulation of starch-positive amyloplasts (red arrowheads) and QC divisions (yellow arrowheads) of the indicated genotypes after mPS-PI-staining of 6-day-old roots. -DEX, mock treated; +DEX, grown for 6 days after germination on 10 µM DEX. c: Percentages of roots with QC division in the indicated genotypes. n.s., not significant by a two-sided Fisher’s exact test. Scale bars: 20 μm.

a-b: Representative confocal images of pWOX5:erCFP/wox5-1 (a) and pWOX5:erCFP 35S:HAN-GR/wox5-1 (b) after 8 h of DEX induction. White dotted lines show QC position. n, numbers of roots analyzed. Scale bars: 20 μm. c: Quantification of the CFP intensity measured in the two central QC cells. In pWOX5:erCFP/wox5-1, n = 13; 35S:HAN-GR pWOX5:erCFP/wox5-1, n = 16 roots were analyzed. Error bars denote SD. p = 1.363 × 10⁻⁶ by a two-sided Student’s t-test. d: Quantification of the pWOX5:H2B-Tdtomato intensity measured in the two central QC cells of the indicated genotypes. Data shown are means of fluorescence intensity from roots of each indicated genotype. Error bars denote SD. In Col-0, n = 24; han-30, n = 36 roots were analyzed. n.s., not significant, by a two-sided Student’s t-test.

a–b: DR5:GFP expression in the Col-0 wild type (a) and pWOX5»bdl (b). The images shown are representative of n = 10 independent roots measured and producing similar results. c-d: QC184 expression in the Col-0 wild type (c) and pWOX5»bdl (d). The images shown are representative of n = 15 independent roots measured and producing similar results. The QC (white) and the CSCs (yellow) are indicated. Scale bars: 50 μm. e-f: Lugol-stained roots of the indicated genotypes in 5-day-old seedlings. Termination of CSCs is indicated by the accumulation of starch granules in wei8-1 tar1-1 mutants (f) compared to the Col-0 wild type (e). Insets show the magnification of CSC niche as indicated by white rectangular. The QC is outlined by dashed lines. White arrowheads indicate the CSC position. g: Frequency of roots with indicated numbers of CSC layers in the indicated genotypes. For Col-0, n = 51, wei8-1, n = 51, tar1-1, n = 60, wei8-1 tar1-1, n = 56 roots were analyzed. n.s., not significant; ***, p < 0.001, comparing presence vs. absence of starch-free CSC layers by a two-sided Fisher’s exact test with Bonferroni correction for multiple testing. p_{Col-0 vs wei8-1 tar1-1} = 0.001. h-i: iCSC induction by WOX5 is largely suppressed in wei8-1 tar1-1 (i), compared with 35S:WOX5-GR in the Col-0 wild-type (h) background. 5-day-old seedlings were induced by 10 μM DEX for 16 h. Dotted white lines indicate QC position. Asterisk with waved brackets indicate iCSC layers. In e-i, Scale bars: 20 μm. j: Quantification of the suppression of extra CSC layers as shown in (h-i). Error bars denote SD of 15 measurements. ****, p < 0.0001 by a two-sided Fisher’s exact test.

a: the coherent feed-forward loop (cFFL) with the input signal s. b: Results of the simulation of the network motif with noisy input. The dashed line denotes the periodic and noisy WOX5 abundance. CDF4 NOR: wiring of the cFFL by a NOR-gate, CDF4 NAND: wiring of the cFFL by a NAND-gate, CDF4 WOX5-only: no HAN inhibition. The parameters are the same for all cases and read: k₁ = k₂ = k₃ = k₄ = k₅ = k₆ = 0.1 h⁻¹, K₁ = K₂ = 6. The signal s(t) into WOX5 is turned off at t = 120 h. To explore the effect of noisy input signals and a loss of signal on the cFFL motif, we modelled the network shown in panel (a) using Ordinary Differential equations: \(\frac{{dx}}{{dt}}={k}_{1}s\left(t\right)-{k}_{2}x\). \(\frac{{dy}}{{dt}}={k}_{3}x-{k}_{4}y\). \(\frac{dz}{dt}={k}_{5}\overrightarrow{{\bf{1}}}\cdot \overrightarrow{{\bf{c}}}-{k}_{6}z\), with x \(\stackrel{\wedge}{=}\) [WOX5], y \(\stackrel{\wedge}{=}\) [HAN], and z \(\stackrel{\wedge}{=}\) [CDF4]. The input signal s is modelled as a log-normally distributed stochastic variable constructed from the stochastic process: \(d\mu \left(t\right)=\alpha \cos \omega t-{\tau }^{-1}\mu (t)+\sqrt{2/\tau \varepsilon {dW}(t)}s(t)={e}^{\mu \left(t\right)-{\varepsilon }^{2}/2}\) with τ = 3 h and ε = 0.2, α = 0.15 h⁻1, ω = 0.26 h⁻¹ (which corresponds to a period of 24 h). Note, that for α = 0 h^-1 μ is an Ornstein-Uhlenbeck process and s has mean 1 and variance \({e}^{{\varepsilon }^{2}}\) – 1. The vector \(\vec{{\boldsymbol{c}}}\) = (c₀₀, c₁₀, c₀₁, c₁₁) represents the four different states of the promoter for CDF4. c₀₀ is the state of free binding sites, that is, neither WOX5 nor HAN is bound, c₁₀ denotes the state of only WOX5 bound, etc. Using a quasi-steady state approximation, we can write for the states: \(\begin{array}{cc}{c}_{00}=\scriptstyle\frac{1}{(1+{K}_{1}x)(1+{K}_{2}y)} & {c}_{10}=\scriptstyle\frac{{K}_{1}x}{(1+{K}_{1}x)(1+{K}_{2}y)}\\ {c}_{01}=\scriptstyle\frac{{K}_{2}y}{(1+{K}_{1}x)(1+{K}_{2}y)} & {c}_{11}=\scriptstyle\frac{{K}_{1}x{K}_{2}y}{(1+{K}_{1}x)(1+{K}_{2}y)}\end{array}\). K₁ and K₂ are the equilibrium constants for the binding of WOX5 and HAN, resp. The NOR-gate logic is given by \(\vec{{\boldsymbol{l}}}\) = (1, 0, 0, 0) and a NAND-gate logic by \(\vec{{\boldsymbol{l}}}\) = (1, 1, 1, 0). The WOX5 only response is modelled via setting K₂ to zero and using the NOR-Gate logic. The results of the simulation can be seen in panel (b). Due to the noisy input signal s(t) WOX5 fluctuates. We compare three different scenarios: i) inhibition by WOX5 only (CDF4 only WOX5), ii) combining the WOX5 and HAN signal in a NOR gate (CDF4 NOR), iii) combining the WOX5 and HAN signal in a NAND gate (CDF4 NAND). In all three cases the motif acts as a low-pass filter, smoothing the response of CDF4. The striking difference between the different wirings is the response to a loss of WOX5: while the WOX5-only and the NAND-gate wiring behave similarly, the NOR-gate wiring shows a delayed response to the decay of WOX5. The NAND-gate exhibits the opposite behaviour; the response is faster compared to the WOX5-only network.

The response of the motif with the different wirings can be observed in Bode plot. The plot is generated by applying an input signal in WOX5 of a specific period and recording the amplitude of the output signal (CDF4). The attenuation of a perturbation with a period of 24 h for the cFFL NOR motif (CDF4 NOR) is approximately twice that of the direct WOX5 attenuation (WOX5 only). Amp.(∞) means the amplitude of the output by constant input. The x-axis denotes the period of the input. The cFFL NOR motif shows the strongest attenuation in particular for input signals with larger periods (long wavelength signals). The parameters are the same as in Extended Data Fig. 8.