Many plant species display remarkable developmental plasticity and regenerate new organs after injury. Local signals produced by wounding are thought to trigger organ regeneration but molecular mechanisms underlying this control remain largely unknown. We previously identified an AP2/ERF transcription factor WOUND INDUCED DEDIFFERENTIATION1 (WIND1) as a central regulator of wound-induced cellular reprogramming in plants. In this study, we demonstrate that WIND1 promotes callus formation and shoot regeneration by upregulating the expression of the ENHANCER OF SHOOT REGENERATION1 (ESR1) gene, which encodes another AP2/ERF transcription factor in Arabidopsis thaliana. The esr1 mutants are defective in callus formation and shoot regeneration; conversely, its overexpression promotes both of these processes, indicating that ESR1 functions as a critical driver of cellular reprogramming. Our data show that WIND1 directly binds the vascular system-specific and wound-responsive cis-element-like motifs within the ESR1 promoter and activates its expression. The expression of ESR1 is strongly reduced in WIND1-SRDX dominant repressors, and ectopic overexpression of ESR1 bypasses defects in callus formation and shoot regeneration in WIND1-SRDX plants, supporting the notion that ESR1 acts downstream of WIND1. Together, our findings uncover a key molecular pathway that links wound signaling to shoot regeneration in plants.

(2015) 'PRC2 represses dedi erentiation of mature somatic cells in Arabidopsis.', Nature plants., 1 (7). p. 15089.Further information on publisher's website:http://dx.doi.org/10.1038/nplants.2015.89Publisher's copyright statement:Additional information: Use policyThe full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-pro t purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details. subscreened for those that initiate ectopic proliferation of mature cells. We reasoned that root hairs represent a suitable system for this study since they have a unicellular structure on the root 3 epidermis with highly specialised functions in water and nutrient uptake 4 . As shown in Fig. 1a (Fig. 1), pointing to the requirement of PRC2 activity in suppressing ectopic proliferation of differentiated cells. Remarkably, sustained divisions ultimately lead to the formation of calli, some of which further develop into somatic embryos that show typical accumulation of lipids (Fig. 1a).Several lines of evidence demonstrate that PRC2 deficiency leads to mitotic reactivation in terminally differentiated root hairs. First, the root hair-specific differentiation marker pEXP7:NLS-GFP 8 shows a similar expression pattern in WT, heterozygous and homozygous emf2-3 vrn2-1 roots, indicating that in the absence of PRC2 root hair differentiation occurs indistinguishably from WT (Fig. 1b). In addition, root hairs of 7-day-old emf2-3 vrn2-1 plants are initially unicellular and only become multicellular in older plants (Fig. 1c). Serial observations of emf2-3 vrn2-1 roots expressing plasma membrane (LTI6-GFP) and nucleus (H2B-YFP) markers 9further indicate that only fully elongated root hairs undergo nuclear and cellular division (Fig. 1d).Multicellularisation of root hairs does not appear to follow a typical gradient along the root axis, suggesting that it is not correlated with the timing of their initial differentiation ( Supplementary Fig. 1). Time-lapse imaging of emf2-3 vrn2-1 root hairs expressing LTI6-GFP and H2B-YFP confirms that these nuclear divisions are accompanied by the formation of a new cell plate and are therefore clearly distinct from the nuclear fragmentation occasionally observed in WT root hairs 10 (Fig. 1e, Supplementary Video 1).As part of the differentiation program, Arabidopsis root hairs undergo several rounds of 4 endoreduplication, a modified cell cycle in which cells replicate nuclear DNA without mitoses and concomitantly increase nucleus and cell size 11 . Entry into the endoreduplication cycle is generally accepted as a commitment for terminal differentiation since cells that have endoreduplicated do not nor...

Callus formation and de novo organogenesis often occur in the wounded tissues of plants. Although this regenerative capacity of plant cells has been utilized for many years, molecular basis for the wound-induced acquisition of regeneration competency is yet to be elucidated. Here we find that wounding treatment is essential for shoot regeneration from roots in the conventional tissue culture of Arabidopsis thaliana. Furthermore, we show that an AP2/ERF transcription factor WOUND INDUCED DEDIFFERENTIATION1 (WIND1) plays a pivotal role for the acquisition of regeneration competency in the culture system. Ectopic expression of WIND1 can bypass both wounding and auxin pre-treatment and increase de novo shoot regeneration from root explants cultured on shoot-regeneration promoting media. In Brassica napus, activation of Arabidopsis WIND1 also greatly enhances de novo shoot regeneration, further corroborating the role of WIND1 in conferring cellular regenerative capacity. Our data also show that sequential activation of WIND1 and an embryonic regulator LEAFY COTYLEDON2 enhances generation of embryonic callus, suggesting that combining WIND1 with other transcription factors promote efficient and organ-specific regeneration. Our findings in the model plant and crop plant point to a possible way to efficiently induce callus formation and regeneration by utilizing transcription factors as a molecular switch.

CRISPR/Cas9 is a programmable nuclease composed of the Cas9 protein and a guide RNA (gRNA) molecule. To create a mutant potato, a powerful genome-editing system was required because potato has a tetraploid genome. The translational enhancer dMac3, consisting of a portion of the OsMac3 mRNA 5′-untranslated region, greatly enhanced the production of the protein encoded in the downstream ORF. To enrich the amount of Cas9, we applied the dMac3 translational enhancer to the Cas9 expression system with multiple gRNA genes. CRISPR/Cas9 systems targeting the potato granule-bound starch synthase I (GBSSI) gene examined the frequency of mutant alleles in transgenic potato plants. The efficiency of the targeted mutagenesis strongly increased when the dMac3-installed Cas9 was used. In this case, the ratio of transformants containing four mutant alleles reached approximately 25% when estimated by CAPS analysis. The mutants that exhibited targeted mutagenesis in the GBSSI gene showed characteristics of low amylose starch in their tubers. This result suggests that our system may facilitate genome-editing events in polyploid plants.

Plant cells have high plasticity for differentiation against various environmental challenges. One of the most striking examples of cellular reprogramming in plants is dedifferentiation of somatic adult cells after wounding, the phenomenon also found in other multicellular organisms.1 Plants often form amorphous mass of dedifferentiated cells at the wound site, and this so-called "callus" not only functions as a coverage but it is also utilized as a source of de novo tissue or organ regeneration. 2 We have recently reported that an AP2/ERF transcription factor WOUND INDUCED DEDIFFERENTIATION 1 (WIND1), also called RAP2. 4,3,4 and its close homologs WIND2-4 are wound responsive and function as positive regulators of cell dedifferentiation in Arabidopsis (Arabidopsis thaliana). 2,5,6 The ectopic overexpression of each of the WIND genes is sufficient to induce callus in Arabidopsis. 6 If WIND genes have conserved biological function among plant species is of great interest, especially considering its potential use for the improvement of tissue culture methods. The WIND1 ortholog in an Arabidopsis relative, a salt cress Thellungiella halophila (ThWIND1-L), is reported to be woundinducible and to have an ability to induce spontaneous callus formation in Arabidopsis.7 However, it is currently unknown how widely molecular entities of WIND genes or signaling pathway regulated by WIND genes is conserved.In order to elucidate how widespread WIND transcription factors are, we first performed database searches for 20 plant species: 12 Dicotyledoneae, 4 Monocotyledoneae, 1 Lycopodiophyta, 1 Bryopsida, and 2 Chlorophyceae (Table S1). Putative WIND orthologs were defined by the result of reciprocal BLAST searches (details are described in Materials and Methods), and they were found in all species we examined except Chlamydomonas reinhardtii and Volvox carteri ( Fig. 1A; Table 1). Green algae are known to have AP2/ERF transcription family, 8 thus WIND subclade likely evolved from AP2/ERF family after the divergence of land plants from green algae. Peptide motif composition is highly conserved in dicotyledons and monocotyledons; however, almost all conserved motifs except the AP2/ERF domain are lacking in the putative orthologs of Selaginella moellendorffii and Physcomitrella patens ( Fig. 1B; Table 1). Thus it is plausible that molecular functions of these orthologs in Magnoliophyta are shared with WIND genes in Arabidopsis.Next, we asked if cellular and developmental response to WIND1 gain-of-function is common in different species. We chose crop species for this experiment, aiming for future applicative research. We have previously shown that 17β-estradiolinduced AtWIND1 promotes callus formation in Arabidopsis. 6 In this study, we extended these observations and found that dexamethasone (DEX)-mediated AtWIND1 induction, namely 35S:AtWIND1-Glucocorticoid Receptor (GR) Arabidopsis treated with DEX, also strongly enhances callus induction on phytohormone-free media ( Fig. 2A and B). We introduced the same DEX-mediated AtWIND1 i...