Control of Arabidopsis apical-basal embryo polarity by antagonistic transcription factors

Abstract

Plants, similarly to animals, form polarized axes during embryogenesis on which cell differentiation and organ patterning programs are orchestrated. During Arabidopsis embryogenesis, establishment of the shoot and root stem cell populations occurs at opposite ends of an apical-basal axis. Recent work has identified the PLETHORA (PLT) genes as master regulators of basal/root fate, whereas the master regulators of apical/shoot fate have remained elusive. Here we show that the PLT1 and PLT2 genes are direct targets of the transcriptional co-repressor TOPLESS (TPL) and that PLT1/2 are necessary for the homeotic conversion of shoots to roots in tpl-1 mutants. Using tpl-1 as a genetic tool, we identify the CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) transcription factors as master regulators of embryonic apical fate, and show they are sufficient to drive the conversion of the embryonic root pole into a second shoot pole. Furthermore, genetic and misexpression studies show an antagonistic relationship between the PLT and HD-ZIP III genes in specifying the root and shoot poles.

Figure 1. Misregulation of PLT genes is necessary for tpl-1 apical to basal transformation

a–c, Seedlings from embryos grown at 29°C. a, WT seedling. b, tpl-1 double root. c, tpl-1 plt1-5 plt2-1 monocot. d–g in situ hybridization with PLT1 and PLT2 antisense probe, embryos grown at 29°C. d, PLT1 expression in WT. e, PLT1 expression in tpl-1. f, PLT2 expression in WT. g, PLT2 expression in tpl-1. h, graph of fold enrichment at the PLT1 locus from ChIP of TPLp::TPL-HA. i, graph of fold enrichment at the PLT2 locus from ChIP of TPLp::TPL-HA. Scale bars, 1 mm (a–c) and 50 µm (d–g).

Figure 2. Molecular characterization of tpl-1, phb-14d, and tpl-1 phb-14d in embryos grown at 29°C

a–f, PHB in situ hybridizations. WT globular (a) and early heart (b) stage, tpl-1 transition stage (c), tpl-1 phb-14d transition stage (d), phb-14d heart stage (e), and phb-1d heart stage (f). g–i, FIL in situ hybridizations. WT (g), tpl-1 (h), and tpl-1 phb-14d (i). j–l, WUS in situ hybridizations. WT (j), tpl-1 (k), and tpl-1 phb-14d. (l). m–p, miR165/166 sensor. GFP fluorescence in WT (m) and tpl-1 (n). GFP in situ hybridizations in WT (o) and tpl-1 (p). q, PHB in situ hybridizations in tpl-1 plt1-5 plt2-1. r, REV in situ hybridization in tpl-1 plt1-5 plt2-1. Arrowheads indicate the root meristem organizing center. Scale bars, 50 µm (a–r).

Figure 3. HD-ZIP III genes antagonize PLT function

a–c, in situ hybridizations with PLT1/PLT2 in 29°C grown embryos. a, PLT1 in tpl-1 phb-14d. b, PLT1 in tpl-1 rev- 10d. c, PLT2 in tpl-1 phb-14d. d, PLT2 in tpl-1 rev-10d. e–h, PLT1/PLT2 in situ hybridizations in 24°C grown embryos. e, PLT1 in tpl-1. f, PLT2 in tpl-1. g, PLT1 in tpl-1 rev-9. h, PLT2 in tpl-1 rev-9. i, phb-14d plt1-5 plt2-1 seedling. j, Scanning electron micrograph (SEM) of rev-10d plt1-5 plt2-1 seedling. k, REV in situ hybridization in rev-10d plt1-2 plt2-1. Scale bars, 50 µm (a–h, k) and 1 mm (i, j).

Figure 4. HD-ZIP III gene misexpression can initiate apical fate and acts antagonistically to PLT gene function

a, SEM image of PLT2p:REVΔmiR-GR seedling. b, PLT2p:PHBΔmiR-GR seedling. c, PLT2p::ICU4ΔmiR-GR seedling. d–h, in situ hybridizations with anti-sense PIN4 probe. d–f, WT 16-cell (d), globular (e), and heart (f) stage. g–h, PLT2p:REVΔmiR-GR globular stage (g) and late heart stage (h) embryos after dexamethasone induction. i–j, in situ hybridizations with anti-sense WUS probe in PLT2p:REVΔmiR-GR globular (i) and heart (j) stage embryos after induction. k–m, in situ hybridizations with anti-sense ANT probe in WT transition stage (k) and PLT2p:REVΔmiR-GR transition stage (l) and torpedo stage (m) embryos after induction. Scale bars, 1 mm (a–c) and 50 µm (d–m).

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