Deciphering the Role of POLYCOMB REPRESSIVE COMPLEX1 Variants in Regulating the Acquisition of Flowering Competence in Arabidopsis^{<a href="#fn1" rid="fn1" class=" fn">1</a>}

Abstract

Polycomb group (PcG) proteins are conserved epigenetic regulators that mediate gene repression through the incorporation of histone-modifying marks (Calonje, 2014). As far as it is known, PcG proteins associate in two multiprotein complexes in Arabidopsis (Arabidopsis thaliana): POLYCOMB REPRESSIVE COMPLEX1 (PRC1) and PRC2. The combined activity of the two complexes is required for stable repression of the target genes.

The major function of PRC2 is to perform histone H3 lysine-27 trimethylation (H3K27me3) through the methyltransferase activity of CURLY LEAF (CLF) and SWINGER (SWN) during sporophyte development or of MEDEA in the endosperm (Chanvivattana et al., 2004). Other PRC2 components are the Drosophila melanogaster suppressor of zeste12 homologs VERNALIZATION2 (VRN2), EMBRYONIC FLOWER2 (EMF2), and FERTILIZATION-INDEPENDENT SEED2, which confer specificity to the resulting PRC2s even though they have some overlapping functions (Chanvivattana et al., 2004), and finally MULTICOPY SUPPRESSOR OF INHIBITORY REGULATOR OF THE RAS-CYCLIC AMP PATHWAY and FERTILIZATION-INDEPENDENT ENDOSPERM, which are common subunits for the different PRC2s (Derkacheva and Hennig, 2014). On the other hand, the identity of Arabidopsis PRC1 is not defined yet. PRC1-mediated function can be histone 2A monoubiquitination (H2Aub) dependent, through the E3 ubiquitin ligase activity of the PRC1 RING finger proteins Arabidopsis B lymphoma Moloney murine leukemia virus insertion region1 homolog 1A (AtBMI1A)/B/C and AtRING1A/B, or H2Aub independent, which requires the activity of the PRC1 component EMF1 (Bratzel et al., 2010, 2012; Yang et al., 2013a; Calonje, 2014). These different PRC1 activities suggest the existence of PRC1 functional variants that may target different subsets of genes (Merini and Calonje, 2015). Another protein used to be considered as a putative PRC1 component is LIKE-HETEROCHROMATIN PROTEIN1 (LHP1), which has the ability to bind H3K27me3 marks (Turck et al., 2007); however, it was recently shown that LHP1 copurifies with PRC2, changing the notion of LHP1 as a PRC1 component (Derkacheva et al., 2013).

From a mechanistic point of view, recent data indicated that the binding and activity of PRC1 are required for H3K27me3 marking at some target genes, which challenges the classical hierarchical model for the recruitment of PcG complexes (Yang et al., 2013a; Calonje, 2014; Merini and Calonje, 2015). Whether this happens at all PcG targets is not yet known. In any case, both PRC1 and PRC2 play important roles in regulating developmental phase transitions in Arabidopsis. For instance, the combined activity of AtBMI1 and PRC2 is crucial for the transition from embryonic to vegetative development (Bratzel et al., 2010; Bouyer et al., 2011; Yang et al., 2013a); EMF1 and PRC2 regulate the transition from vegetative to reproductive development (Sung et al., 1992; Kinoshita et al., 2001; Schubert et al., 2006); and AtRING1A was recently shown to be involved in the regulation of several flowering repressors, suggesting its participation in the transition to flowering (Shen et al., 2014). However, thus far, little is known about the implication of PcG proteins in another important developmental change, the transition from juvenile to adult phase that marks the acquisition of reproductive competence.

Following germination, plants pass through a phase of vegetative growth that can be further divided into a juvenile and an adult vegetative phase. During the juvenile-to-adult phase transition, plants acquire competence to flower as well as undergo changes in multiple traits, such as leaf size and shape, internode length, and trichome distribution (Huijser and Schmid, 2011; Poethig, 2013). Although PcG proteins may have a role in regulating this developmental transition, the severity of the phenotype in some PcG mutants or the lack of phenotype in others has concealed their possible implication. Conversely, two microRNAs (miRNAs), miR156 and miR172, and their targets have been identified as key components of the mechanisms that underlie juvenile-to-adult phase changes. The miR156 targets transcripts of a subset of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) transcription factors that have been shown to promote the transition from juvenile to adult and to flowering (Wu and Poethig, 2006; Schwarz et al., 2008). By contrast, miR172 targets APETALA2 (AP2)-like factors that have been shown to repress both the transition to flowering and flower development (Aukerman and Sakai, 2003; Schmid et al., 2003; Jung et al., 2007; Mathieu et al., 2009). The expression of these miRNAs is temporally regulated by age; thus, as the plant ages, miR156 levels decrease, resulting in an increase in SPL expression. In the shoot apical meristem (SAM), the SPL proteins activate the floral pathway integrators SUPPRESSOR OF CONSTANS1 (SOC1) and AGAMOUS-LIKE24 (AGL24) and the floral meristem identity genes FRUITFULL, LEAFY (LFY), and AP1; and in leaves, the SPLs activate miR172 expression that in turn down-regulates the AP2-like floral repressors, which inhibit the floral integrator FLOWERING LOCUS T (FT; Wang, 2014). The so-called age pathway is proposed to prevent flowering during the juvenile phase and ensure plant flowering even in the absence of exogenous inductive cues.

FT, in addition to being regulated by the age pathway, is strongly controlled by photoperiod; in fact, the level of FT expression at the end of long days plays a primary role in determining when Arabidopsis flowers (Turck et al., 2008; Wigge, 2011). The circadian clock sets a high CONSTANS (CO) mRNA expression in the late afternoon in long days, which coincides with light exposure, resulting in CO protein accumulation as light stabilizes the CO protein. The vasculature-expressed CO protein promotes FT expression activation in the phloem companion cells, specifically at the end of long days (Imaizumi and Kay, 2006; Turck et al., 2008). During the night, CO is rapidly degraded by the proteasome and FT expression is repressed (Valverde et al., 2004). Upon its production at dusk, the FT protein moves from phloem to the SAM, where it interacts with the locally transcribed FLOWERING LOCUS D (FD) transcription factor to activate floral integrators like SOC1 and AGL24 to induce flowering (Amasino, 2010; Matsoukas et al., 2012). Accordingly, genetic studies have placed the age pathway parallel with the photoperiodic pathway (Wang, 2014), both being required to determine the threshold of FT necessary for flowering competence.

Several direct regulators of miR172-encoding genes have been identified, including the MADS box factor SHORT VEGETATIVE PHASE, which downregulates the levels of miR172 (Cho et al., 2012), GIGANTEA, which mediates the photoperiod activation of miR172 (Jung et al., 2007), and SPL9, which leads to an accumulation of miR172 (Wu et al., 2009). On the other hand, recent evidence indicates that the seed maturation gene FUSCA3 (FUS3) contributes to the direct expression of the primary transcripts of MIR156A and MIR156C (pri-MIR156A and pri-MIR156C) in the developing seed and that this expression is important after germination to delay the juvenile-to-adult vegetative phase transition (Wang and Perry, 2013). However, upstream effectors mediating the age-dependent decline in miR156 levels are largely unknown. Interestingly, several recent studies showed a correlation between plant nutritional status and miR156 levels. The accumulation of metabolically active sugars, such as Suc and Glc, acts as a signal to selectively repress the expression of the miR156A and miR156C genes (Wahl et al., 2013; Yang et al., 2013b; Yu et al., 2013), but the molecular mechanism by which this repression take place and is maintained is not yet understood.

In this work, we show that loss of function of the PRC1 component AtBMI1 leads to the up-regulation of pri-MIR156A/C at the time the levels of miR156 should decline, resulting in an extended juvenile phase and delayed flowering. We found that atbmi1a/b mutants display reduced levels of H3K27me3 marks at the transcriptional start site (TSS) of these genes, suggesting the participation of the PcG machinery in regulating miR156 expression. According to our results, AtBMI1-mediated repression of pri-MIR156A/C allows the age-dependent expression of FT and the development of adult traits. Interestingly, the PRC1 component EMF1 does not regulate pri-MIR156A/C expression; instead, EMF1 participates in the regulation of miR172. Our findings show how the combined regulatory roles of two functional PRC1 variants are crucial to coordinate the acquisition of flowering competence.

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