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

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

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

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

BRR2a Affects Flowering Time via <i>FLC</i> Splicing

<div><p>Several pathways control time to flowering in <i>Arabidopsis thaliana</i> through transcriptional and posttranscriptional gene regulation. In recent years, mRNA processing has gained interest as a critical regulator of flowering time control in plants. However, the molecular mechanisms linking RNA splicing to flowering time are not well understood. In a screen for Arabidopsis early flowering mutants we identified an allele of <i>BRR2a</i>. BRR2 proteins are components of the spliceosome and highly conserved in eukaryotes. Arabidopsis BRR2a is ubiquitously expressed in all analyzed tissues and involved in the processing of flowering time gene transcripts, most notably <i>FLC</i>. A missense mutation of threonine 895 in BRR2a caused defects in <i>FLC</i> splicing and greatly reduced <i>FLC</i> transcript levels. Reduced <i>FLC</i> expression increased transcription of <i>FT</i> and <i>SOC1</i> leading to early flowering in both short and long days. Genome-wide experiments established that only a small set of introns was not correctly spliced in the <i>brr2a</i> mutant. Compared to control introns, retained introns were often shorter and GC-poor, had low H3K4me1 and CG methylation levels, and were often derived from genes with a high-H3K27me3-low-H3K36me3 signature. We propose that BRR2a is specifically needed for efficient splicing of a subset of introns characterized by a combination of factors including intron size, sequence and chromatin, and that <i>FLC</i> is most sensitive to splicing defects.</p></div

Intron retention in <i>brr2a</i>-2 is associated with specific chromatin properties.

<p>Box plots show averaged genome-wide bisulfite sequencing and ChIP signals for exons (blue) and introns (green). DRI, differentially retained introns in <i>brr2a</i>-2. DRI gene, genes containing at least one differentially retained intron. Thus, the boxes in each plot represent, from left to right, (i) exons from genes that have no DRIs, (ii) exons from genes that have at least one DRI, (iii) normally spliced introns in genes that have at least one DRI, (iv) DRIs, and (v) introns from genes that have no DRIs. Control genes without DRIs were selected to have the same median expression as the DRI genes. Significant differences are indicated; numbers are–log₁₀ of p-values from Wilcoxon signed-rank tests.</p

Developmental alterations in <i>cäö</i>.

<p>(A) Silhouette of the sixth rosette leaf from wild type (Col, left) and <i>cäö</i> (right) showing the serrated margin of the <i>cäö</i> leaf. Plants were grown for 4 weeks under LD conditions. (B) Rosette morphology of Col and <i>cäö</i> plants at time of bolting in LD; scale bar: 5 cm. (C) Leaf morphology of Col and <i>cäö</i> in 20 days old plants; scale bar: 1 cm. (D) Reduced silique length in <i>cäö</i> mutants. Scale bar: 1 mm (E) Cleared wild type (left panel) and <i>cäö</i> ovules with arrested (middle) or absent female gametophytes (right panel). Scale bar: 25 μm. Cells of the egg apparatus are indicated: CC, central cell; EC, egg cell; SY, synergids.</p

Intron retention in <i>brr2a</i>-2.

<p>(A) Number of alternative splicing events (AS) based on three biological replicates each of Col and <i>brr2a</i>-2. ES, exon skipping; AA, alternative acceptor site; AD, alternative donor site; IR, intron retention; C, complex, a combination of one or several of ES, AA, AD or IR. The increased number of AS events in <i>brr2-a</i> is mainly due to increased intron retention. (B) Number of complex alternative splicing events. 2IR, two intron retention events in the same transcript; IRIR, either one of two different introns is retained; 3IR, three intron retention events in the same transcript; 2IRIR, either a pair of introns or a more 3' located single intron is retained; IR2IR, either a single intron or a more 3' located pair of introns is retained; 4IR, four intron retention events in the same transcript. For (A) and (B), AS events were quantified from RNA-seq data using ASTALAVISTA [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005924#pgen.1005924.ref049" target="_blank">49</a>]. The complex AS events observed in <i>brr2a</i>-2 are mainly combinations of IR events. (C) Schematic representation of basic AS events. (D) Total intron number per transcript with detected IR (left) and number of retained introns per transcript with detected IR (right). Whereas transcripts with detected IR have on average 6 transcripts, only one or two of those are retained, suggesting that IR is mostly an intron and not a transcript property. (E) Length of introns with increased retention in <i>brr2a</i>-2 (“retained”), with decreased retention (“released”) and with no change in retention (“equal”). Introns with increased retention are often shorter whereas introns with decreased retention are often longer than unaffected introns. (F) GC content of introns with increased retention in <i>brr2a</i>-2 (“retained”), with decreased retention (“released”) and with no change in retention (“equal”). Introns with increased retention have often lower GC content whereas introns with decreased retention have often higher GC content than unaffected introns. P-values are from one-sided t-tests.</p