Reprogramming of H3K27me3 Is Critical for Acquisition of Pluripotency from Cultured Arabidopsis Tissues

Abstract

In plants, multiple detached tissues are capable of forming a pluripotent cell mass, termed callus, when cultured on media containing appropriate plant hormones. Recent studies demonstrated that callus resembles the root-tip meristem, even if it is derived from aerial organs. This finding improves our understanding of the regeneration process of plant cells; however, the molecular mechanism that guides cells of different tissue types to form a callus still remains elusive. Here, we show that genome-wide reprogramming of histone H3 lysine 27 trimethylation (H3K27me3) is a critical step in the leaf-to-callus transition. The Polycomb Repressive Complex 2 (PRC2) is known to function in establishing H3K27me3. By analyzing callus formation of mutants corresponding to different histone modification pathways, we found that leaf blades and/or cotyledons of the PRC2 mutants curly leaf swinger (clf swn) and embryonic flower2 (emf2) were defective in callus formation. We identified the H3K27me3-covered loci in leaves and calli by a ChIP–chip assay, and we found that in the callus H3K27me3 levels decreased first at certain auxin-pathway genes. The levels were then increased at specific leaf genes but decreased at a number of root-regulatory genes. Changes in H3K27me3 levels were negatively correlated with expression levels of the corresponding genes. One possible role of PRC2-mediated H3K27me3 in the leaf-to-callus transition might relate to elimination of leaf features by silencing leaf-regulatory genes, as most leaf-preferentially expressed regulatory genes could not be silenced in the leaf explants of clf swn. In contrast to the leaf explants, the root explants of both clf swn and emf2 formed calli normally, possibly because the root-to-callus transition bypasses the leaf gene silencing process. Furthermore, our data show that PRC2-mediated H3K27me3 and H3K27 demethylation act in parallel in the reprogramming of H3K27me3 during the leaf-to-callus transition, suggesting a general mechanism for cell fate transition in plants.

A transcriptional view on somatic embryogenesis

“…Organogenesis from leaf explants involves several H3K27me3‐dependent events. First, loss of H3K27me3 marks results in early upregulation of auxin pathway genes, followed by repression of leaf identity gene expression via increased H3K27me3, and finally by root meristem gene expression (He, Chen, Huang, & Xu, 2012). PRC2 function is not required for callus formation from roots; therefore PRC2 seems to be required to eliminate leaf identity by silencing leaf‐specific genes.…”

Auxin-Independent NAC Pathway Acts in Response to Explant-Specific Wounding and Promotes Root Tip Emergence during de Novo Root Organogenesis in Arabidopsis

“…S2C). We conducted qRT-PCR analyses to test whether naphthylacetic acid (NAA; a type of auxin) could induce the transcription of the auxin response gene GH3.2 (Staswick et al, 2005;He et al, 2012) and NAC1. The results showed that the NAA treatment quickly induced GH3.2 transcription by 2 h after culture (Fig.…”

“…Transgenic plants were obtained by Agrobacterium tumefaciens-mediated transformation of each of these constructs into Col-0. WOX11 pro :GUS, WOX5 pro :GUS, and DR5 pro :GUS transgenic plants were described previously (Ulmasov et al, 1997;He et al, 2012;Liu et al, 2014). The primers used for plasmid construction are listed in Supplemental Table S3.…”

“…The RNA extractions and qRT-PCR analyses were performed as previously described (He et al, 2012) using gene-specific primers. The qRT-PCR results are shown as relative expression levels normalized against those produced using Actin-specific primers.…”

“…In tissue culture, adventitious roots and shoots can regenerate from a group of pluripotent cell mass, termed callus. Callus is usually induced by a high level of auxin in the medium, and callus formation was shown to follow the rooting developmental pathway (Sugimoto et al, 2010;He et al, 2012;Liu et al, 2014).…”