2,4-D treatment is required to trigger the development of a functional root apical meristem.

<p>(A) Expression of <i>PLT1</i> and <i>PLT2</i> in <i>clf swn</i> and wild-type (WT) somatic embryos (SE), SE-like (SE-L) structures, zygotic embryos (ZE) and seedlings. (B) Expression of shoot (<i>pCLV3</i>::<i>GUS</i>) and root (<i>pWOX5</i>::<i>NLS-GUS</i>) apical meristem markers in WT-derived ZEs and SEs and in <i>clf swn</i> 2,4-D-induced SEs and mock SE-L structures. (C) Germination efficiency of SEs and SE-L structures after 7 days on hormone-free medium. Examples of germinated <i>clf swn</i> SEs or SE-L structures are shown on the right. Scale bars: white = 2 mm, unlabeled black = 0.2 mm. Bars in graphs represent means ±SEM (N = 2 biol. replicates, 40 embryos each). H-F—hormone-free medium, seedl–seedling, h—hour, d–day, DAG–days after germination.</p

PRC2 Represses Hormone-Induced Somatic Embryogenesis in Vegetative Tissue of <i>Arabidopsis thaliana</i>

<div><p>Many plant cells can be reprogrammed into a pluripotent state that allows ectopic organ development. Inducing totipotent states to stimulate somatic embryo (SE) development is, however, challenging due to insufficient understanding of molecular barriers that prevent somatic cell dedifferentiation. Here we show that Polycomb repressive complex 2 (PRC2)-activity imposes a barrier to hormone-mediated transcriptional reprogramming towards somatic embryogenesis in vegetative tissue of <i>Arabidopsis thaliana</i>. We identify factors that enable SE development in PRC2-depleted shoot and root tissue and demonstrate that the establishment of embryogenic potential is marked by ectopic co-activation of crucial developmental regulators that specify shoot, root and embryo identity. Using inducible activation of PRC2 in PRC2-depleted cells, we demonstrate that transient reduction of PRC2 activity is sufficient for SE formation. We suggest that modulation of PRC2 activity in plant vegetative tissue combined with targeted activation of developmental pathways will open possibilities for novel approaches to cell reprogramming.</p></div

16 transcription factor genes upregulated specifically in the wounded and 2,4-D-treated <i>clf swn</i> shoot apex (<i>cs</i>-WA).

<p>16 transcription factor genes upregulated specifically in the wounded and 2,4-D-treated <i>clf swn</i> shoot apex (<i>cs</i>-WA).</p

2,4-D induces somatic embryo (SE) development in PRC2-depleted tissue.

<p>(A) 7-day 5 μM 2,4-D treatment of <i>clf swn</i> PRC2 mutants but not wild-type (WT) or single mutant shoot explants results in SE development. (B) 60-hour exposure to 5 μM 2,4-D is sufficient to induce efficient SE formation in <i>clf swn</i>. Following 2,4-D treatment, callus forms in WT (C) while SEs develop from <i>clf swn</i> (D). Stunted embryos form in <i>clf swn</i> after long (7-day) exposure to 2,4-D (E). (F–T) Morphology of explants: (F–I) <i>clf swn</i> explants on hormone-free medium. (J–L) <i>clf swn</i> explants treated with 2,4-D. (M–O) WT SEs originating from 2,4-D-treated ZEs. Generally, SEs do not develop after 2,4-D treatment of <i>clf swn</i> root (P), cotyledon or true leaf (Q) and hypocotyl (R). Occasionally, SE development on cotyledon margins was observed (S). No visible SE formation is observed at the end of a 7-day 2,4-D treatment of the <i>clf swn</i> shoot apex before transfer to hormone-free medium (T). Black arrowheads in (D, E, L, N) indicate the site of most frequent SE attachment to parental explant. Grey arrowheads in (H, I) point to vascular tissue attaching the SE-like structures to parental explants. Embryonic lipids (G, K, O, S) were visualized by Sudan Red 7B. Bars in graphs represent means ±SEM (N = 3–4 biol. replicates with 50 (A) or 30 (B) explants each). Red number above bars in (A) and (B) indicate the percentage of SE formation ±SEM in mock (DMSO)-treated explants. Scale bars: white = 2 mm, black = 0.2 mm. H-F -hormone-free medium, h—hour, d—day.</p

The potential to form somatic embryos (SE) from zygotic embryos (ZE) is lost during germination of wild-type Arabidopsis.

<p>Immature early bent cotyledon stage ZEs (A) are exposed to 5 μM 2,4-D (B) followed by transfer to hormone-free medium where SEs form (D, E). (C vs. D, F) 7 days of 2,4-D are required for efficient reprogramming to SE. (G) The potential to form SEs is reduced in dry seeds and lost during germination. Scale bars: (A, B) = 0.5 mm, (C, D) = 2 mm. Bars in F, G represent means ±SEM (F: N = 3 biol. replicates, >70 ZEs each; G: N = 4, >90 seeds each). H-F—hormone-free medium, d—day.</p

External ABA induces expression of shoot and embryonic regulators in <i>clf swn</i> roots.

<p>Expression of developmental marker genes in wild-type (WT) and <i>clf swn</i> explants exposed to different treatments for 60 hours: (A) ABA-responsive genes <i>ABI3</i>, <i>ABI4;</i> and <i>PLT5/EMK</i>; (B) root-identity genes <i>PLT1</i>, <i>PLT2</i> and <i>WOX5</i>; (C) SE-inducing genes <i>PLT4/BBM</i>, <i>AGL15</i> and <i>WUS</i>; (D) shoot/embryo-identity genes <i>CUC1</i>, <i>CUC2</i> and <i>DRNL</i>. Bars represent mean ±SEM (N = 2 biol. replicates). Orange bars represent tissue samples from which embryos can develop. A—2,4-D (auxin), M—mock, W—wounding, B—ABA.</p