Plant stem cell maintenance involves direct transcriptional repression of differentiation program

Abstract

In animal systems, master regulatory transcription factors (TFs) mediate stem cell maintenance through a direct transcriptional repression of differentiation promoting TFs. Whether similar mechanisms operate in plants is not known. In plants, shoot apical meristems serve as reservoirs of stem cells that provide cells for all above ground organs. WUSCHEL, a homeodomain TF produced in cells of the niche, migrates into adjacent cells where it specifies stem cells. Through high-resolution genomic analysis, we show that WUSCHEL represses a large number of genes that are expressed in differentiating cells including a group of differentiation promoting TFs involved in leaf development. We show that WUS directly binds to the regulatory regions of differentiation promoting TFs; KANADI1, KANADI2, ASYMMETRICLEAVES2 and YABBY3 to repress their expression. Predictions from a computational model, supported by live imaging, reveal that WUS-mediated repression prevents premature differentiation of stem cell progenitors, being part of a minimal regulatory network for meristem maintenance. Our work shows that direct transcriptional repression of differentiation promoting TFs is an evolutionarily conserved logic for stem cell regulation.

Figure 1

System for assaying WUS-response genes at higher spatial resolution. (A) A schematic of Arabidopsis shoot apical meristem (SAM) stem cell niche showing stem cell domain/the central zone (CZ), the organizing center (OC)/niche and differentiating region/the peripheral zone (PZ). Transcriptional domains of WUS, CLV3 and WUS protein gradient are highlighted in different colors. Mutual feedback regulation between CLV3 and WUS is shown. (B, C) Three-dimensional reconstructed top views of ap1-1;cal1-1 SAMs carrying Dex-inducible form of WUS (35S:;WUS-GR), labeled with stem cell marker-pCLV3::mGFP-ER (green) in plants treated with mock and Dex, respectively, are shown. Arrows point to outer limits of stem cell domain and expansion of stem cell domain upon Dex treatment. Scale bar in (C) represents 25 μm and it remains same for (B). (D) qRT–PCR analysis showing temporal dynamics of CLV3 activation upon WUS induction. Error bars represent standard deviation for two biological replicates.

Figure 2

A spatial map of WUS-responsive genes. (A, C) Genes upregulated by Dexamethasone (Dex)-inducible WUS in the absence and presence of Cycloheximide (Cyc), respectively. Genes downregulated by WUS in the absence (B) and presence (D) of Cyc. Numbers on the CZ, the PZ and the RM refer to the number of WUS-responsive genes that are expressed in each domain. Numbers indicated in between cell types are the WUS-responsive genes that are expressed in two adjacent cell types. Normalized gCRMA values (using multiexperiment viewer (MEV4) software) showing relative expression levels of representative set of genes activated (E) and repressed (G) by WUS in various treatments shown on x axis. Expression profiles of WUS-regulated genes shown in (E) and (G) were confirmed by real-time qRT–PCR, relative expression levels for various treatments indicated on x axis are shown for activated (F) and repressed (H) genes. Profile plots for few genes in (E) are not visible as these genes share similar expression profiles as others.

Figure 3

Differentiation promoting transcription factors are direct transcriptional targets of WUS. (A–D) Chip-qPCR showing relative enrichment of regulatory regions of KAN1, KAN2, AS2 and YAB3, respectively. Error bars represent standard deviation. Genomic regions are mapped with respect to transcription start site (+1). (E) EMSA showing recombinant WUS protein bound to radiolabeled-oligonucleotides corresponding to KAN1, KAN2, AS2 and YAB3 regulatory regions. A black arrowhead indicates free probe and a dark gray arrowhead band shift for KAN1, KAN2, AS2 and YAB3 radiolabeled oligonucleotides. respectively. The sequences of WUS bound oligonucleotides are shown in (F) and conserved TAAT elements are highlighted. (G) EMSA showing recombinant WUS protein bound to radiolabeled KAN1−1100 and KAN1 1050 oligonucleotides and in the absence and presence of anti-WUS antibody. A black arrowhead indicates free probe, a dark gray arrowhead indicates band shift for KAN1−1100 and KAN1 1050 and a white arrowhead indicates ‘super shift’.

Figure 4

TAAT elements are required for WUS binding and repression. (A–G) EMSAs showing recombinant WUS protein bound to radiolabeled oligonucleotides corresponding to KAN1, KAN2, AS2 and YAB3 regulatory regions and mutant oligonucleotides versions of conserved TAAT sequences. A black arrowhead indicates free probe and a dark gray arrowhead band shift for KAN1, KAN2, AS2 and YAB3 radiolabeled oligonucleotides, respectively. The sequences of WUS bound oligonucleotides and mutants are shown in (H). All TAAT elements are underlined and TAAT elements that are essential for WUS binding are shown in bold letters. (I) The TAAT promoter element is essential for WUS-dependent repression of KAN1 in Arabidopsis leaf mesophyll protoplasts. Transient transfection assay plots showing repression of LUCIFERASE (LUC) reporter when cloned downstream of a region containing WUS-binding element found in KAN1 promoter (KAN1 +950, +1150:35s::LUC) and a mutated version (TGGT) of KAN1 promoter. The constructs were tested for transactivation of LUC by cotransfection with or without WUS. The cotransfection of UBIQUITIN::GUS served as an internal control. Activity was expressed as a ratio of firefly LUC/GUS activity. Three biological replicates were used for each experiment and the error bars represent the standard deviation.

Figure 5

Design and optimization of the computational model. (A) Illustration of the transcriptional interactions in the model mapped on typical functional domains/cell types of SAMs. (B) The 229 parameter sets from the optimization procedure, displayed using principal component analysis (first and second principal components: e₁, e₂). The color scale represents the variation of equilibrium KAN1 expression between the wild type and a perturbation (transient ubiquitous CLV3 expression). The gray disk is centered at the parameter set used in all spatial example simulations (Supplementary Table 8). In (C–F), first column shows Confocal side views of SAMs showing cell boundaries (red) and RNA or protein distribution domains (green). Corresponding gene names are given on each panel. Scale bar shown in (C) represents 25 μm (same for C–F). Second column: Templates used for optimization showing corresponding gene expression patterns (green). Third column: Simulation output from the example optimized parameter set. The color scale label ‘normal’ indicates the template defined gene expression level. (C–E) Expression patterns, templates and optimized model for WUS (taken from Yadav et al, 2009), CLV3 (taken from Reddy and Meyerowitz, 2005) and KAN1, respectively. (F) Distribution of WUS protein in the SAM (taken from Yadav et al, 2011) and in the model. The color scale for WUS concentration in the model is capped at the concentration value of WUS repressing KAN1 to half its maximal expression (k_w/K).

Figure 6

Dynamics of reorganization of stem cells and differentiating progenitors upon transient manipulation of WUS levels. (A–D) 3D-reconstructed top views of SAMs labeled with stem cell/CZ -pCLV3::mGFP5-ER (endoplasmic reticulum localized GFP) and the pKANADI1::KANADI1:GFP (nuclear localized GFP) expressed in differentiating cells located at the outer edges of the PZ. Plasma membrane localized YFP highlights outlines of all cells (red). Scale bar shown in (A) represents 25 μm and it remains same for (A–D). Arrows in all panels point to KAN1 expressing cells. (A, B) A time-lapse series showing pCLV3 and KAN1 expression before (A) and 24 h after (B) Dex-mediated overexpression of WUS (35S::WUS:GR). Note expansion of pCLV3 along with loss of pKAN1 expression (B). (C, D) A time-lapse series showing pCLV3 and pKAN1 expression prior to (C) and 96 h after (D) Dex-mediated overexpression of CLV3 (35S::GR:LhG4; 6XOP::CLV3), a complete time-lapse series is given in Supplementary Figure 9. Note a progressive shift of KAN1 expression toward the receding central zone (D). Time-course evolution of modeled CLV3 (E, G) and KAN1 (F, H) expression upon transient ubiquitous expression of WUS (E, F) or CLV3 (G, H). Note that (G) shows both native and induced CLV3 expression, and the native promoter activity is decreasing due to the loss of WUS. Time points (t) are fractions of the time from perturbation induction to model stabilization (T). The color scale label ‘normal’ indicates the template defined gene expression level. Upon WUS overexpression, a substantial increase in CLV3 and decrease in KAN1 expression domains are observed in both experiment and simulation. Upon CLV3 overexpression, KAN1 expression domain extends toward the CZ in both experiment and simulation. Additional model perturbations are displayed in Supplementary Figure 3.