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RESEARCH ARTICLE

Molecular Plant 7, 977–988, June 2014

A Histone H3 Lysine-27 Methyltransferase Complex Represses Lateral Root Formation in Arabidopsis thaliana Xiaofeng Gua,b, Tongda Xub,c, and Yuehui Hea,b,c,1 a Department of Biological Sciences, National University of Singapore, Singapore b Temasek Life Sciences Laboratory, Singapore c Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

ABSTRACT  Root branching or lateral root formation is crucial to maximize a root system acquiring nutrients and water from soil. A lateral root (LR) arises from asymmetric cell division of founder cells (FCs) in a pre-branch site of the primary root, and FC establishment is essential for lateral root formation. FCs are known to be specified from xylem pole pericycle cells, but the molecular genetic mechanisms underlying FC establishment are unclear. Here, we report that, in Arabidopsis thaliana, a PRC2 (for Polycomb repressive complex 2) histone H3 lysine-27 (H3K27) methyltransferase complex, functions to inhibit FC establishment during LR initiation. We found that functional loss of the PRC2 subunits EMF2 (for EMBRYONIC FLOWER 2) or CLF (for CURLY LEAF) leads to a great increase in the number of LRs formed in the primary root. The CLF H3K27 methyltransferase binds to chromatin of the auxin efflux carrier gene PIN FORMED 1 (PIN1), deposits the repressive mark H3K27me3 to repress its expression, and functions to down-regulate auxin maxima in root tissues and inhibit FC establishment. Our findings collectively suggest that EMF2–CLF PRC2 acts to down-regulate root auxin maxima and show that this complex represses LR formation in Arabidopsis. Key words:  lateral root formation; founder cell establishment; PRC2; PIN1; auxin maximum. Gu X., Xu T., and He Y. (2014). A histone H3 lysine-27 methyltransferase complex represses lateral root formation in Arabidopsis thaliana. Mol. Plant. 7, 977–988.

Introduction The root system largely consists of the primary root and lateral roots (LRs). LRs are formed iteratively in an acropetal manner in the differential zone along the primary root. Root branching or LR formation is critical for maximizing a root system to acquire nutrients and water from soil, and under complex control by endogenous factors such as the phytohormone auxin and environmental factors such as nutrients and/or water availability (Casson and Lindsey, 2003; Benkova and Bielach, 2010; Petricka et al., 2012). A lateral root arises from asymmetric cell division of founder cells (FCs) at a pre-branch site of the primary root, and FC establishment is essential for LR formation. FCs are specified from xylem pole pericycle (XPP) cells that are primed to be competent for FC specification in the basal meristem just above the root tip (Benkova and Bielach, 2010; De Smet, 2012). FC priming involves self-sustained oscillations of auxin responsiveness and gene expression in the primary root oscillation zone above the root tip, and auxin plays a critical role for priming XPP cells (De Smet et al., 2007; Moreno-Risueno et al., 2010).

An auxin-response maximum in the protoxylem cell file of basal meristem has been proposed to prime neighboring XPP cells for FC specification, and auxin accumulation in the XPP cells is one of the earliest events in FC establishment (Dubrovsky et  al., 2008; De Smet, 2012). Auxin is perceived by an F-box protein receptor in a ubiquitin ligase complex, and promotes interaction of the receptor with the AUXIN /INDOLE-3-ACETIC ACID (Aux/ IAA) proteins that heterodimerize with AUXIN RESPONSE FACTOR (ARF) transcription factors to repress their activities; the auxin-mediated interaction targets an Aux/IAA for proteasome-dependent degradation, and thus frees ARFs for transcriptional activation of auxin-responsive genes (Chapman and Estelle, 2009; Benkova and Bielach, To whom correspondence should be addressed. E-mail dbshy@ nus.edu.sg, tel. +65 6872-7978, fax +65-6872-7007. © The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/ssu035, Advance Access publication 7 April 2014 Received 8 September 2013; accepted 25 March 2014 1

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2010). Recent studies in Arabidopsis have revealed that FC priming requires the IAA28 (for INDOLE-3-ACETIC ACID28)-dependent auxin signaling (De Rybel et  al., 2010). This signaling controls the expression of GATA TRANSCRIPTION FACTOR 23 (GATA23) in XPP cells, which is required for FC specification. Besides GATA23, other genes directly involved in FC establishment remain to be identified. Local auxin maxima and gradients in root tissues result predominantly from cell-to-cell polar auxin transport (Petrasek and Friml, 2009). Auxin is transported from shoot towards root tip via the vascular cambium, and subsequently is re-directed to lateral cells and tissues. In Arabidopsis, the PIN-FORMED (PIN) family efflux carriers located at the plasma membrane mediate the polar auxin transport (PAT) in the primary root and LRs as well, and consist of PIN-FORMED 1 (PIN1), PIN2, PIN3, PIN4, and PIN7, among which the basal membrane-localized PIN1 in the stele plays a major role in transporting auxin towards the root tip (Petrasek and Friml, 2009). The abundance of PIN proteins plays an important role in the regulation of auxin transport. PIN expression is positively feedback regulated by auxin and requires PLETHORA genes (Blilou et al., 2005; Vieten et al., 2005; Chen et al., 2011). Recently, it has been shown that auxin up-regulates the expression of a MADSbox transcription factor that directly promotes PIN1 and PIN4 expression (Garay-Arroyo et  al., 2013). To date, the genes (if any) that directly repress PIN expression in roots remain elusive. Covalent modifications of histone tails regulate eukaryotic gene expression. The evolutionarily conserved Polycomb group (PcG) proteins function to repress developmental gene expression and thus regulate developmental processes in plants and animals (Schuettengruber et al., 2007). In Arabidopsis, the four core subunits of the animal PcG complex PRC2 (for Polycomb repressive complex 2) including two structural components known as Esc and Nurf55, the Zinc-finger protein Suppressor of zeste 12 (Su(z)12), and Enhancer of zeste (E(z), a histone H3 lysine-27 (H3K27) methyltransferase) are well conserved, and their homologs form several PRC2-like complexes (Butenko and Ohad, 2011). For instance, EMBRYONIC FLOWER2 (EMF2, a Su(z)12 homolog), CURLY LEAF (CLF, an E(z) homolog), MULTICOPY SUPPRESSOR OF IRA1 (a Nurf55 homolog), and FERTILIZATION INDEPENDENT ENDOSPERM (the Esc homolog) together form an EMF–PRC2 complex that plays multiple critical roles during vegetative development (Butenko and Ohad, 2011; Bemer and Grossniklaus, 2012). PRC2s catalyze repressive H3K27 trimethylation (H3K27me3) mainly in genic regions including proximal promoter regions and gene bodies, to repress the expression of thousands of genes in the Arabidopsis genome, the majority of which are developmental genes (Zhang et al., 2007; Bemer and Grossniklaus, 2012).

Arabidopsis root growth and development involve chromatin-based mechanisms. In the root tip, the slowdividing quiescent center (QC) and surrounding stem cells form the root stem-cell niche (SCN) (Petricka et al., 2012). The histone acetyltransferase known as GCN promotes the expression of root stem-cell transcription factors to maintain SCN (Kornet and Scheres, 2009). An Arabidopsis H3K4 methyltransferase SET DOMAIN GROUP 2 is also required for SCN maintenance and functions to promote the growth of primary root and LRs (Yao et al., 2013); in contrast, the CLF H3K27 methyltransferase functions to inhibit the stemcell activity in the primary root (Aichinger et  al., 2011). Moreover, an ATP-dependent CHD3 chromatin remodeler, PICKLE, has been shown to be necessary for the expression of several root stem-cell transcription factors and for the maintenance of stem-cell activity (Aichinger et  al., 2011). Additionally, histone deacetylation has been shown to be involved in the formation of root cortex and root hairs (Cui and Benfey, 2009; Liu et al., 2013). To date, the chromatin mechanisms, if any, that mediate the FC establishment and LR initiation remain elusive. Here, we report that an EMF–CLF PRC2 complex inhibits FC establishment during LR initiation. We show that the CLF H3K27 methyltransferase directly binds to PIN1 chromatin, deposits H3K27me3 to repress PIN1 expression, and functions to down-regulate auxin maxima in root cells and tissues, leading to proper root growth and development.

INTRODUCTION RESULTS The EMF–PRC2 Subunit EMF2 Functions to Inhibit Arabidopsis Primary Root Growth and LR Formation EMF2, encoding an animal Su(z)12 homolog, plays an essential role in proper growth and development in Arabidopsis (Moon et al., 2003; Kim et al., 2010). Loss-offunction emf2 mutants skip vegetative phase and produce small inflorescence upon germination (Moon et al., 2003). To understand the biological role of EMF2 for vegetative development, we explored a vascular-specific promoter, the widely used SUC2 (SUCROSE TRANSPORTER 2) promoter (Truernit and Sauer, 1995), to knock down EMF2 expression in vascular tissues such as leaf veins and the root vascular cylinder using a double-stranded RNA interference (dsRNAi) approach with an EMF2-specific fragment (Figure  1A). Two independent transgenic lines homozygous for a single T-DNA locus were created: EMF2– RNAi-1 and EMF2–RNAi-2. At seedling stage, these lines, compared to the parental line Col, exhibited curly leaves and longer primary roots (Figure 1B and 1C). Subsequently, we quantified EMF2 transcript levels in the roots of these RNAi lines and found that indeed EMF2 expression was

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Figure 1  EMF2 Knockdown Causes Increased Primary Root Growth and LR Formation. (A) EMF2 gene structure. Exons are represented by black boxes; the blue bar indicates the 204-bp region used to knock down EMF2 expression. (B) Phenotypes of EMF2-knockdown lines (single-locus T3 homozygotes). Shown are 10-day-old seedlings. (C) Primary root length of the indicated seedlings. 20–23 10-day-old seedlings were scored for each line; error bars indicate standard deviation (SD). (D) Relative EMF2 transcript levels in the roots of indicated seedlings. The transcripts were quantified by RT–qPCR, and normalized to the endogenous control TUBULIN2 (TUB2); relative expression to Col is presented; error bars for SD of three measurements. (E) LR number per primary root of the indicated seedlings. 20–23 10-day-old seedlings were scored for each line; bars for SD. (F, G) LRP number per primary root (F) and LRP density (G) of the indicated seedlings. 20–21 10-day-old seedlings were scored for each line; bars for SD. (C, E–G) Double asterisks indicate statistically significant differences (p