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WUSCHEL protein movement mediates stem cell homeostasis in the Arabidopsis shoot apex - PubMed

  • ️Sat Jan 01 2011

WUSCHEL protein movement mediates stem cell homeostasis in the Arabidopsis shoot apex

Ram Kishor Yadav et al. Genes Dev. 2011.

Abstract

WUSCHEL (WUS) is a homeodomain transcription factor produced in cells of the niche/organizing center (OC) of shoot apical meristems. WUS specifies stem cell fate and also restricts its own levels by activating a negative regulator, CLAVATA3 (CLV3), in adjacent cells of the central zone (CZ). Here we show that the WUS protein, after being synthesized in cells of the OC, migrates into the CZ, where it activates CLV3 transcription by binding to its promoter elements. Using a computational model, we show that maintenance of the WUS gradient is essential to regulate stem cell number. Migration of a stem cell-inducing transcription factor into adjacent cells to activate a negative regulator, thereby restricting its own accumulation, is a theme that is unique to plant stem cell niches.

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Figures

Figure 1.
Figure 1.

WUS protein movement. (A) A sketch of the Arabidopsis shoot apex showing various cell types compared with domains of WUS RNA and protein distribution. 3D reconstructed top view of a wus-1 mutant SAM rescued by pWUS::eGFP-WUS (B) and FMs of stage 2 (C), stage 3 (C), and stage 5 (D) showing eGFP-WUS protein (in green); cell outlines were stained with FM4-64 (red). (E,F) Side views of SAMs showing eGFP:WUS protein extending into the PZ and into the L1 layer. (G,H) Side views of early and late stage FMs showing WUS protein localization. I, J, and K are side views of SAMs, early flowers, and late flowers, respectively, showing eGFP mRNA distribution in pWUS::eGFP:WUS-expressing plants. Bar, 20 μM.

Figure 2.
Figure 2.

WUS protein movement is required for its function. WUS protein localizations of pWUS::eGFP:WUS (A–F), pWUS::nls-GFP:WUS (G–L), and pWUS::2XGFP:WUS (M–R). (S–V) pWUS::eGFP:WUS in clv3-2 mutants. (Green) WUS protein; (red) cell membranes. A, G, M, and S are 3D reconstructed top views of SAMs. C, I, and O are confocal cross-sections showing the L1 layer of SAMs. B, E, F, H, K, L, N, Q, R, T, and V are reconstructed side views of SAMs and FMs. (FM3) Stage 3 flower; (FM4) stage 4 flower); (FM5) stage 5 flower. Arrows point to the L1 layer (L1) and the L2 layer (L2). Opposing arrows in B, H, and N indicate the width of WUS protein domains. Arrowheads in D, J, and P indicate the radial domain of WUS expression in FM3. Bars: C,I,O, 10 μM; all other panels, 35 μM.

Figure 3.
Figure 3.

CLV3 is a direct transcriptional target of WUS. (A) qRT–PCR showing the increase in CLV3 transcripts upon activation of WUS by using a Dex-inducible system within 2 h of Dex application in the absence and presence of protein synthesis inhibitor Cyc. Error bars represent standard deviation. (B) ChIP-qPCR showing relative enrichment of regulatory regions of CLV3. Regions are mapped with respect to transcription start site (+1). (C) The clv3-3 T-DNA allele is located 55 nt in front of the WUS-binding elements located downstream from the ORF. (D, left panel, lanes 1–6) EMSA showing recombinant WUS protein bound to radiolabeled CLV3 oligonucleotides from the +970 and +997 downstream regions and from the −1080 upstream region. (Middle panel, lanes 7–12) Absence of binding of WUS to mutant forms of CLV3 (−1080)-radiolabeled oligonucleotides in which each of the four bases (in red) within the TAAT core have been substituted. (Right panel, lanes 13–16) EMSA showing recombinant WUS protein bound to radiolabeled CLV3 oligonucleotides −1080 in the absence and presence of anti-WUS antibody A black arrowhead indicates free probe, a gray arrowhead indicates band shift, and a white arrowhead indicates “supershift.” The sequences of wild-type and mutant form (M1–M4) oligonucleotides are indexed in E. (F) Transient transfection assay plots showing activation of LUC reporter when cloned downstream from a 35S minimal promoter region containing the WUS-binding element found in the CLV3 promoter [CLV3(−1000,−1200):35s::LUC] and a region containing WUS-binding elements found in the CLV3 downstream region [CLV3(+900,+1100):35s::LUC].

Figure 4.
Figure 4.

A computational model for 3D patterning of gene expressions in the SAM. (A) Illustration of the gene interactions in the model showing typical expression domains in the SAM. (B) Simulation results from a CLV3–WUS feedback model (see the Supplemental Material). The top row shows WUS expression, and the bottom row shows CLV3 expression. The left panel shows the wild-type expression domains, the middle panel shows the results from a perturbed WUS diffusion (the disappearance of the CLV3 expression can be interpreted as a terminated meristem), and the right panel shows the expanded stem cell domain/CLV3 expression domain along the outer layers of cells upon misexpression of WUS from the CLV3 promoter, as shown in earlier studies (Brand et al. 2002; Yadav et al. 2010). In the color bar, a linear scale is used and “normal” represents the template value used for optimization (1; i.e., concentration value 1 in arbitrary units).

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