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Plant, Cell and Environment (2014) 37, 1259–1269

doi: 10.1111/pce.12236

Review

Type-II histone deacetylases: elusive plant nuclear signal transducers Vincent Grandperret, Valérie Nicolas-Francès, David Wendehenne & Stéphane Bourque

Pôle Mécanisme et Gestion des Interactions Plantes-microorganismes – ERL CNRS 6300, Université de Bourgogne, UMR 1347 Agroécologie, 17 rue Sully, BP 86510, Dijon cedex 21065, France

ABSTRACT Since the beginning of the 21st century, numerous studies have concluded that the plant cell nucleus is one of the cellular compartments that define the specificity of the cellular response to an external stimulus or to a specific developmental stage. To that purpose, the nucleus contains all the enzymatic machinery required to carry out a wide variety of nuclear protein post-translational modifications (PTMs), which play an important role in signal transduction pathways leading to the modulation of specific sets of genes. PTMs include protein (de)acetylation which is controlled by the antagonistic activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Regarding protein deacetylation, plants are of particular interest: in addition to the RPD3-HDA1 and Sir2 HDAC families that they share with other eukaryotic organisms, plants have developed a specific family called type-II HDACs (HD2s). Interestingly, these HD2s are well conserved in plants and control fundamental biological processes such as seed germination, flowering or the response to pathogens. The aim of this review was to summarize current knowledge regarding this fascinating, but still poorly understood nuclear protein family. Key-words: nucleus; post-translational modifications; signal transduction.

INTRODUCTION For a long time, the nucleus has solely been considered as the organelle where replication and transcription occur. This restricted view was largely due to the sparse knowledge about the numerous functions of the nucleus. For instance, it was widely acknowledged that molecules with a molecular weight below 40 kDa could freely diffuse through the nuclear pores (Fedorenko et al. 2010). However, it is now clearly established that the transport of ions and molecules across the nuclear envelope is strictly controlled, regardless of their molecular weight. This was nicely exemplified with calcium by Pauly et al. (2000). In their study, the authors used intact nuclei extracted from transformed tobacco cells that expressed the calcium reporter aequorin. They demonstrated Correspondence: Stéphane Bourque. E-mail: stephane.bourque @dijon.inra.fr © 2013 John Wiley & Sons Ltd

that the nucleus could perceive by itself changes in its environment – in this case a sorbitol-induced hyperosmotic stress – and convert that information into variations in free Ca2+ concentrations. The way Ca2+ diffusion is controlled is still unknown at the moment.This finding implies that the nucleus possesses proper molecular machineries that allow for the generation of specific signalling pathways. Thus, the nucleus is a major actor in the determination of the specificity of the cellular response (Vaahtera & Brosché 2011). Such a role is also based on the assumption that the nucleus displays two major properties: firstly it is thought to contain the enzymatic arsenal required for mediating signal transduction; secondly it is thought to harbour control systems that regulate the translocation of molecular signals, regardless of their chemical nature (Vaahtera & Brosché 2011). Indeed, even if the functioning of the nuclear pores is not yet fully understood (Tamura & Hara-Nishimura 2011), several recent studies have pointed out some interesting properties (Meier & Somers 2011). For example, in response to light, phytochromes are translocated from the cytosol into the nucleus, a signalling step necessary for the regulation of light-controlled genes. More precisely, when exposed to light, phytochrome B (phyB) undergoes conformational changes that reveal a functional nuclear localization signal (NLS) allowing for its translocation into the nucleus (Chen et al. 2005). Regarding other phytochromes that lack an NLS such as phytochrome A (phyA), they are translocated through their interaction with the protein far-red elongated HYpocotyl 1 (FHY1), which contains an NLS (Genoud et al. 2008). Once a specific signal is translocated into the nucleus, it initiates pathways that result in the regulation of the expression of specific sets of genes that mediate the proper response. Importantly, components of these pathways are also subjected to post-translational modifications (PTMs). Despite the fact that PTMs are highly diversified (more than 300 PTMs have been described so far; Jensen 2004) and affect a large number of proteins (over one-third of human proteins could be regulated through phosphorylation; Zolnierowicz & Bollen 2000), so far only a few studies have investigated PTMs in nuclear signalling (see Dahan et al. 2011b, for a recent review). Furthermore, although protein (de)phosphorylation is certainly the most studied PTM in the plant cell nucleus (specific phosphoproteomics approaches are now devoted to nuclear proteins; Jones et al. 2009), the mode of action of the protein kinases and phosphatases involved in nuclear signalling is still largely unknown (Dahan 1259

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et al. 2010). Nuclear protein (de)acetylation is another PTM that plays a major role in the regulation of gene expression (Chen & Tian 2007). In this regard, several studies highlight the key involvement of histone deacetylases (HDACs) in signalling processes. These proteins catalyse deacetylation not only of histones, but also of non-histone proteins potentially involved in signal transduction. Interestingly, plants possess an exclusive class of HDACs called type-II HDACs (HD2s). HD2s show little structural similarity with the other two HDAC families that plants share with other eukaryotes. In this review, we describe the characteristics of this exclusive plant HDAC family with special emphasis on their regulation and contribution to plant cell responses to biotic and abiotic stresses.

Molecular characteristics of plant HDACs The PTM of a protein through the chemical binding of an acetyl group from acetyl-S∼CoA to the amine function of Lys residues was firstly described for histones (Allfrey et al. 1964). Once identified, the enzymes that catalyse that process were named histone acetyltransferases (HATs; Gallwitz 1971). Conversely, HDACs were named based on their ability to remove the acetyl group. However, because these enzymes also act on proteins unrelated to histones (e.g. transcription factors or coregulators of gene transcription), HATs and HDACs are now usually referred to as lysine acetyl transferases and lysine deacetylases, respectively (Chen & Tian 2007). Interestingly, depending on their isoforms, both HATs and HDACs have been localized mainly in the plant cell nucleus, confirming that (de)acetylation could be a critical process in the regulation of nuclear processes. Since their first identification, plant HDACs have been classified according to their similarity with other eukaryotic HDACs, particularly with yeast HDACs (Pandey et al. 2002). Two major HDAC families are common to plants and other eukaryotic organisms: the RPD3 (reduced potassium dependency protein 3) – HDA1 (HDAC 1) superfamily and the SIR2 (silent information regulator 2) family. The third family, HD2, is specific to plant cells and strictly diverges from the other families, at least at the structural level (Fu et al. 2007). Table 1 illustrates current knowledge of the molecular features of the three HDAC families in the model plant Arabidopsis thaliana.

The RPD3-HDA1 superfamily: The first plant HDAC belonging to the RPD3-HDA1 superfamily – ZmRpd3 – was identified in maize and was found to functionally complement a yeast rpd3 mutant (Rossi et al. 1998). In A. thaliana, among the 18 genes encoding proteins with deacetylase activity, 12 HDAC homologs of ZmRPD3 were identified and classified into three different classes based on clustering patterns and bootstrap supports: Class I (composed of HDA6, 7, 9, 10, 17 and 19 – also called HDA1) defines the RPD3 group, Class II (including HDA5, 15 and 18) defines the HDA1-like group and Class IV (composed of only one member, HDA2) defines the AtHDA2 group

(Alinsug et al. 2009, 2012). It is important to note that the latter class is also termed Class III in some other studies (e.g. Hollender & Liu 2008). The other RPD3 HDACs – namely HDA8 and HDA14 – remain unclassified. Although all of these proteins have a typical deacetylase domain located in their N-terminal part, they exhibit distinct structural features that could reflect differences in terms of activity (Table 1). Notably, depending on the isoform, RPD3-HDA1 proteins contain NLS and/or NES (nuclear export signal) motifs or not in their sequences. In fact, in mammals, HDACs are known to be subjected to nucleocytoplasmic shuttling. This process constitutes an original mode of regulation of their activity that consists in sequestering HDACs in compartments away from their nuclear substrates (Shen et al. 2006). In plants, this is exemplified by class II HDACs that are located either in the nucleus or the cytosol, depending for example on varying environmental conditions. Such is the case of HDA15, which is delocalized from the nucleus to the cytosol in response to light sensing (Alinsug et al. 2012). This shuttling process can easily be explained by the presence of predicted NLSs and/or NESs in numerous class II HDACs. Regarding functions, among the 12 members of the RPD3/ HDA1 family only two – namely HDA6 and HDA1/HDA19 – have been extensively studied. HDA6 plays a role in many fundamental processes such as transcriptional gene silencing (Probst et al. 2004), flowering and senescence (Wu et al. 2008) or ABA (abscisic acid) and salt signalling (Chen et al. 2010). At the molecular level, using an RNAi knockdown approach, Earley et al. (2006) showed that HDA6 can deacetylate the lysine residues of various substrates including histones H3 (K5 and K14) and H4 (K12). Since HDA6 is localized in the nucleolus (Pontes et al. 2007), deacetylation of nucleolar histones likely silences rRNA genes. HDA1/HDA19 was reported to regulate embryonic development (Tanaka et al. 2008) and responses to both biotic and abiotic stresses (Song et al. 2005; Chen & Wu 2010; Choi et al. 2012). The mode of action of HDA1/HDA19 could diverge from that of HDA6 as it interacts with a non-histone protein, LEUNIG, a member of the GroTLE transcription corepressor family (Gonzalez et al. 2007). However, the molecular targets of all these HDACs remain largely unknown.

The SIR2 family SIR2-like HDACs were grouped based on their sequence homology to the yeast silent information regulator 2 (Sir2) protein. Contrary to RDP3-HDA1 HDACs, SIR2-like HDACs do not need to bind Zn2+ to be active. Thus, they are not inhibited by trichostatin A (TSA) or sodium butyrate unlike RPD3s or HD2s (Jung 2001; Grozinger & Schreiber 2002). However, they display a NAD (nicotinamide adenine dinucleotide)-dependent enzymatic activity: during the catalytic process, the acetyl group is removed from the target protein and transferred to NAD+ (Dali-Youcef et al. 2007). The sirtuins from all organisms are divided into five classes based on sequence motifs within their highly conserved Sir2 domain (Frye 2000; Imai et al. 2000). Arabidopsis has two sirtuin proteins, SRT1 and SRT2, belonging to Classes IV and

© 2013 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 1259–1269

© 2013 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 1259–1269

364 aa

AT5G35600

AT1G08460

AT3G44680

AT3G44660

AT4G33470

AT3G18520

AT3G44490

AT5G61070

AT5G55760

AT5G09230

HDA7

HDA8

HDA9

HDA10

HDA14

HDA15

HDA17

HDA18

SIR2 family Sir1

AT5G22650

AT5G03740

AT2G27840

HDT2 (HD2B)

HDT3 (HD2C)

HDT4 (HD2D)

203 aa

294 aa

306 aa

245 aa

682aa

158aa

552aa

423 aa

142aa

426aa

377 aa

409 aa

471 aa

II

I

II

Unclassified

I

I

Unclassified

I

I

II

IV (or III)

I

Class

Nucleus

Nucleus

Nucleus

Nucleus

Nucleus

Nucleus Cytosol

Nucleus

Nucleus Cytosol

Mitochondria Cytosol

Nucleus

Nucleus

Cytoplasm

Nucleus

Nucleus Chloroplast envelope

Cytoplasm Nucleus ER

Nucleus ?

Nucleus

Localization

Deacetylase domain Large acidic domain Zinc finger NLS Deacetylase domain NLS Deacetylase domain Zinc finger NLS Deacetylase domain NLS

Zinc finger NAD+-deacetylase Zing finger NAD+-deacetylase

Monopartite NLS (13–43) NES Deacetylase (36–328) No NLS NES Deacetylase (93–349) Monopartite NLS (369–381) Bipartite NLS (367–400) No NES Deacetylase (46–348) Monopartite NLS (14–37) Bipartite NLS (15–44 ; 155–182) NES Deacetylase (38–332) Bipartite NLS (149–177) NES Deacetylase (29–323) No NLS NES Deacetylase (39–335) No NLS NES Deacetylase (24–317) No NLS No NES Deacetylase (14–33) No NLS No NES Deacetylase (80–387) NLS NES Deacetylase (171–461) Zinc finger (86–115) No NLS No NES Deacetylase (9–49) NLS NES Deacetylase (79–381)

Structural domains

ABA and salt stress signalling Seed dormancy

ABA and salt stress signalling Seed dormancy

Seed dormancy

Seed dormancy

Plant defence

?

Differentiation of hair and non-hair cells in the root epidermis

Unknown

Negative control of chlorophyll synthesis Enhances Agrobacterium transformation

Association with PP2A-A2 phosphatase and microtubules

Unknown

Flowering

Unknown

Unknown

Jasmonate and salt signalling Senescence Flowering Transcriptionnal gene silencing

Binds 14-3-3 proteins Involved in light response.

Unknown

Repression of salicylic acid synthesis

Function

Luo et al. 2012b

Sridha & Wu 2006; Colville et al. 2011; Luo et al. 2012b

Yano et al. 2013

Colville et al. 2011; Zhou et al. 2004

Wang et al. 2010

–

Xu et al. 2005; Alinsug et al. 2012; Liu et al. 2013a

–

Alinsug et al. 2012; Crane & Gelvin 2007; Liu et al. 2013b

Alinsug et al. 2012; Tran et al. 2012

–

Kim et al. 2013

Alinsug et al. 2012

Alinsug et al. 2009

Probst et al. 2004; Wu et al. 2008; Chen et al. 2010

Alinsug et al. 2012

Alinsug et al. 2012

Zhou et al. 2005; Tanaka et al. 2008; Choi et al. 2012

Ref.

Experimentally confirmed localization. Protein identification is deduced from the TAIR database (http://www.arabidopsis.org). NESs were predicted using NetNes 1.1 Server (http://www.cbs.dtu.dk/services/NetNES/). NLSs were predicted using cNLS Mapper (nls-mapper.iab.keio.ac.jp/cgi-bin/ NLS_Mapper_form.cgi). Other functional domains were predicted using MyHits (myhits.isb-sib.ch/cgi-bin/motif_scan) and ProDom (prodom.prabi.fr/prodom/current/html/home.php) algorithms. ABA, abscisic acid; HD2, type-II histone deacetylases; HDAC, histone deacetylases; HDT, histone deacetylase; NAD, nicotinamide adenine dinucleotide; NES, nuclear export signal; NLS, nuclear localization signal; RPD3-HDA1, reduced potassium dependency protein 3 histone deacetylases 1; PP2A-A2, protein phosphata 2A-A2; SIR2, silent information regulator 2.

AT3G44750

HD2 family HDT1 (HD2A)

Sir2

473 aa

AT5G63110

HDA6 (RPD3B)

660 aa

AT5G61060

HDA5

387 aa

319 aa

AT5G26040

AT4G38130

RPD3-HDA1 family HDA1 (HDA19)

Size

HDA2

Accession

Name

Table 1. Main features of Arabidopsis thaliana HDACs

HD2 as new nuclear signal transducers 1261

Dinocarpus longan

Hordeum vulgare

Oryza sativa

Data reported in this table only concern HD2s studied at the protein and/or functional level. AA, amino acids; CK2, casein kinase 2; HD2, type-II histone deacetylases; HDT, histone deacetylase; NES, nuclear export signal; NLS, nuclear localization signal

6 7 8 4 8 5 7 6 6 9 5 8 134–154 Asp 144–198 Asp/Glu 139–196 Asp/Glu 133–154 Asp 150–196 Asp 99–198 Asp/Glu 99–198 Asp/Glu 131–174 Asp 98–197 Asp/Glu 161–208 Asp – 166–209 Asp Yes – Yes – Yes Yes Yes Yes Yes Yes Yes Yes – – – – Yes Yes Yes – – Yes Yes Yes Yes Yes Yes Yes NLS Yes Yes NLS NLS Yes Yes Yes 245 306 294 203 307 295 294 274 297 310 386 305 Zea mays Nicotiana tabacum

N-terminal peptide Accession Name

Table 2. Characterized plant HD2 proteins

• Almost all HD2s contain the pentapeptide MEFWG in their N-terminal end (Fig. 2). Mass spectrometry analyses showed that in tobacco cells, the N-terminal Met of HD2s is removed (Bourque et al. 2011). It is then interesting to consider the second amino acid of the MEFWG motif, glutamic acid.According to the N-terminal end rule (Gonda et al. 1989),the presence of a glutamic acid at the N-terminal end is associated with a relatively short half-life.Thus, HD2s most probably have a short half-life that participates in the control of their biological functions. This was confirmed with Nicotiana tabacum HD2s whose half-lives are strongly shortened in response to biotic stresses (V. Grandperret and S. Bourque, unpublished data). • The N-terminal part of HD2s contains the deacetylase catalytic domain. It is usually admitted that two wellconserved charged amino acids, namely a histidine residue at position 25 and a glutamic acid residue at position 69, are fundamental to deacetylase activity (Dangl et al. 2001). However, new alignments with HD2 N-terminal deacetylase domains from several complete sequenced plant genomes (Fig. 2) show that the H25 residue in

Deacetyl. domain

Current knowledge about HD2s is scarce, certainly because they are only present in plant cells.According to all sequenced genomes currently available, HD2s belong to small gene families that are composed of two (rice or grapevine) to four (Arabidopsis) members (Table 2). Most of these HD2s have similar architectures (Fig. 1), with conserved domains:

5–97 2–95 1–100 1–95 1–91 1–96 1–96 2–80 5–92 1–91 7–94 1–95

AA

Plant HD2 molecular characteristics

MEFWG MEFWG MEFWG MEFWG MEFWG MEFWG MEFWG MEFWG MEFWG MEFWG MTENHRFWG MEFWG

NLS

NES

C2H2 Zn2+-finger

Acidic domain

CK2 phospho sites

Ref.

The type-II HDAC family is exclusive to plants and shows very little similarity with the other HDAC families (Pandey et al. 2002). The currently known HD2s display similarities with the FKBP family of peptidyl-prolyl cis-trans isomerases (Aravind & Koonin 1998; Dangl et al. 2001). They also display similar organization: a conserved (M)EFWG motif at their N-terminal end, followed by a deacetylase catalytic domain in their N-terminal region, an acidic domain in their central region involved in the regulation of the enzymatic activity and, in their C-terminal region, an NLS and a zinc finger probably involved in protein–protein or protein–DNA interactions. Similarly to the RPD3 family members, Zn2+ binding could be a prerequisite for HD2 activity. However, to our knowledge, no one has so far reported direct evidence that plant HD2s have a real deacetylase activity. There is only indirect evidence: mutation or inhibition of HD2s in rice and tobacco result in the accumulation of histones or nuclear proteins in hyperacetylated forms (Bourque et al. 2011; Ding et al. 2012). According to the literature, these proteins are related to plant stress responses (Sridha & Wu 2006; Colville et al. 2011; Luo et al. 2012b) and seed development (Wu et al. 2000; Yano et al. 2013).

AT3G44750 AT5G22650 AT5G03740 AT2G27840 NP_001105631 ACZ54945 ACZ54946 AAW57802 AAU10714 ACD50315 ACD50316 ?

The type-II HDAC family

HDT1 HDT2 HDT3 HDT4 HDT1 HD2a HD2b HDT702 HDT701 HvHDAC2-1 HvHDAC2-2 DlHD2

II, respectively. Interestingly, SRT2 has at least five splicing variants (Frye 2000; Pandey et al. 2002).

Zhou et al. 2004 Yano et al. 2013 Luo et al. 2012b Luo et al. 2012b Lusser et al. 1997 Bourque et al. 2011 Bourque et al. 2011 Fu et al. 2007 Fu et al. 2007; Ding et al. 2012 Demetriou et al. 2009 Demetriou et al. 2009 Kuang et al. 2012

V. Grandperret et al.

Arabidopsis thaliana

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Figure 1. Schematic representation of plant conserved type-II histone deacetylases (HD2) domains. This schematic structure of plant HD2s is based on AtHDT1. MEFWG, conserved pentapeptide in the N-terminal end; NLS, nuclear localization signal; P, putative casein kinase 2α-specific phosphorylation site.

AtHDA1 is part of a larger conserved HxSQxxL motif. Another conserved domain, PQxsxDlvlxxefElSH including D69, is found at the C-terminal end of the catalytic domain. The biological relevance of these domains is still unknown at the moment. Interestingly, the deacetylase domain of HD2s does not significantly match with that of the other HDAC families, suggesting that these different families could have different target proteins. • All HD2s studied so far possess a well-characterized NLS domain and some of them also contain a NES according to algorithms such as NetNES (http://www.cbs.dtu.dk/ services/NetNES/) or NESsential (seq.cbrc.jp/NESsential/). In accordance with these observations, several studies demonstrate that HD2s are mainly localized in the nucleus (Zhou et al. 2004; Sridha & Wu 2006) and, for some of them, more precisely in the nucleolus (Lusser et al. 1997; Earley et al. 2006). However, to our knowledge, the regulation of these proteins through nucleocytoplasmic shuttling has not been reported yet. • A large central acidic domain containing mostly Asp residues is found in almost all HD2s. This domain is experimentally known to modify the apparent molecular mass of HD2 proteins in SDS-PAGE gels (Brosch et al. 1996; Armstrong & Jones 2003; Bourque et al. 2011). Interestingly, these acidic domains contain Ser and/or Thr residues predicted to be phosphorylated by casein kinase 2α (CK2α). CK2α is a well-known nuclear protein kinase that controls many fundamental biological processes such as the cell cycle, cell differentiation or apoptosis (Litchfield 2003) through the phosphorylation of many different target proteins, including HDACs in mammalian cells

(see for example Tsai & Seto 2002). Four genes in A. thaliana encode CK2α proteins that are located in the nucleus, with the exception of CK2αcp, which is located into the chloroplast (Salinas et al. 2006). Lusser et al. (1997) demonstrated that a human CK2α can phosphorylate ZmHD2, an HD2 from maize, in vitro. However, the molecular consequences of the phosphorylation of HD2s on their enzymatic activity, location or stability are unknown, as well as the identity of the Ser or Thr residues that undergo CK2αinduced phosphorylation. In mammals, phosphorylation can have opposite effects depending on the affected HDAC. Indeed, Tsai & Seto (2002) and Pluemsampant et al. (2008) showed that phosphorylation of HDAC2 (a human HDAC belonging to the RPD3 family) by CK2α activates it in hypoxia-associated tumours. Conversely, phosphorylation of HDAC4 and HDAC5 leads to their translocation from the nucleus to the cytosol where they are sequestered by 14-3-3 proteins (Grozinger & Schreiber 2000). • Around 60% of the HD2s known so far contain a Zn2+ finger in their C-terminal part. These Zn2+ fingers are of the C2H2-type and more precisely belong to the TFIIIAtype (Ciftci-Yilmaz & Mittler 2008). They usually allow for proteins to bind specific DNA sequences through a conserved QALGGH sequence (Isernia et al. 2003; Klug 2010). However, none of the HD2s studied so far contains that sequence, suggesting that the Zn2+ finger could be involved in protein–protein interactions rather than in protein–DNA interactions. We may therefore wonder which are HD2s’ partners. Two studies provide first answers. Firstly, in A. thaliana, Luo et al. (2012b) demonstrated that HD2c interacts with HDA6, an RPD3-type

Figure 2. Alignment of the type-II histone deacetylases (HD2) deacetylase domains from four fully sequenced plant species. This alignment was constructed using the Muscle algorithm present on Mobyle portal (http://mobyle.pasteur.fr) using the four HD2 sequences from Arabidopsis thaliana (accession numbers: AtHD2a, AT3G44750; AtHD2b, AT5G22650; AtHD2c, AT5G03740; AtHD2d, AT2G27840), the three sequences from Medicago truncatula (accession numbers: XP_003606267; AFK45592; XP_003606266), the two sequences from Vitis vinifera (accession numbers: XP_002277422; XP_002270966) and the three sequences from Populus trichocarpa (accession numbers: XP_002313645; XP_002328078; XP_002326411). © 2013 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 1259–1269

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HDAC, and binds to histone H3 to regulate the expression of ABI1 and ABI2, two ABA-responsive genes. Secondly, using yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays, Kuang et al. (2012) showed that DIHD2 physically interacts with DIERF1 – an ethylene-responsive factor – to regulate fruit senescence in longan fruit (Dimocarpus longan). However, in both cases, the involvement of the Zn2+ finger in protein–protein interactions was not investigated. We could also assume a more speculative function: Singh et al. (1999) report that the Zn2+ finger of RanBP2, which is also of the C2H2-type, interacts with exportin-1 (CRM1), which in turn promotes its export from the nucleus to the cytosol. A similar function might be considered for HD2 Zn2+ fingers.

Plant HD2 expression Using expression data collected in the framework of the 1kp-project (http://www.onekp.com/project.html), it becomes clear that HD2s are globally expressed in the entire plant kingdom, from mosses to monocots and dicots. According to the few more detailed reports on HD2 expression published so far in Arabidopsis, rice and tobacco (Zhou et al. 2004; Bourque et al. 2011; Ding et al. 2012), HD2s appear to be expressed in the whole plant, from the roots to the flowers and fruit, with decreased expression in older tissues. However, the relative expression of one isoform or another seems highly variable. For instance, in tobacco leaves, NtHD2c is 40-fold underexpressed as compared with NtHD2a or NtHD2b (S. Bourque, unpublished data). Interestingly, HD2 expression is altered in response to various biotic and abiotic stresses. Indeed, OsHDT701 expression is up-regulated in the compatible interaction with Magnaporthe oryzae isolate Che86, but down-regulated in the incompatible reaction with M. oryzae isolate C9240 (Ding et al. 2012). Furthermore, Li et al. (2011) showed that the expression of OsHDT1 – another denomination of OsHDT701 – displays a circadian rhythm and affects rice flowering. These results suggest that the transcriptional regulation of OsHDT701/ OsHDT1 expression is a main regulatory process in its biological activity. The expression of the four A. thaliana HD2s is also significantly down-regulated in response to ABA (Sridha & Wu 2006; Luo et al. 2012b). Similarly, in barley, the expression of two HD2 genes, HvHDAC2-1 and HvHDAC2-2, is significantly modified in response to ABA treatment, with distinct patterns: whereas HvHDAC2-2 is up-regulated after 24 hours’ treatment, HvHDAC2-2 is down-regulated after 6 hours (Demetriou et al. 2009). Furthermore, using two different cultivars, HvHDAC2-1 was found up-regulated during seed development while HvHDAC2-2 was down-regulated. Thus, it seems difficult to predict the expression profile of a given HD2 as it appears to be highly variable depending on the plant species and/or the HD2 isoform. Interestingly, a comparison of HD2 expression in A. thaliana and in rice using Genevestigator (Hruz et al. 2008) shows that contrary to the two Oryza sativa HD2 isoforms (Fig. 3b), all four A. thaliana HD2 isoforms are similarly regulated in response to biotic stresses (Fig. 3a). Furthermore, contrary to what

happens with ABA, other hormones such as salicylic acid, methyl jasmonate and ethylene do not affect HD2 gene expression according to Genevestigator analyses on Arabidopsis data. However, this analysis also reveals that the amplitude of expression varies according to the isoform, raising the question of the redundancy between the different isoforms in given species.

Biological functions controlled by plant HD2s In the last decade, numerous studies have reported the involvement of HD2s in the control of biological processes, including seed physiology and development, biotic stresses and abiotic stresses.

HD2s in the control of seed dormancy and germination The first study that showed the involvement of histone (de)acetylation in the control of seed dormancy was conducted by Tai et al. (2005). Using a pharmacological approach, they demonstrated that when A. thaliana seeds are treated with TSA, an HDAC inhibitor, dozens of genes involved in seed germination are deregulated, including genes encoding LEA (late embryogenesis abundant) proteins. Subsequently, several reports highlighted the involvement of HD2s in the control of seed dormancy. Bond et al. (2009) showed that nicotinamide, a specific inhibitor of the SIR2 class of HDACs, negatively regulates the expression of the flowering locus C involved in vernalization, confirming that changes in histone acetylation levels in the vicinity of specific gene promoters could be of major importance for seed development. However, HD2s were only recently shown to participate, like the other HDAC classes, in seed development. Using hd2a, hd2c and hd2a/hd2c mutants, Colville et al. (2011) demonstrated that HD2a and HD2c have antagonistic effects on seed germination: HD2a negatively controls while HD2c positively controls seed germination. Interestingly, the germination rate of the hd2a/hd2c double mutant seeds was similar to that of wild-type seeds. Similar results were previously reported for HD2c (Sridha & Wu 2006). Yano et al. (2013) provide evidence that, similarly to HD2a and HD2c, HD2b can control seed germination in A. thaliana. Using 117 Arabidopsis ecotypes with various degrees of seed dormancy collected all around the world and compiling genome-wide association mappings and transcriptome analyses, the authors showed that seed dormancy was strongly associated with AtHD2b expression. Indeed, suppressing its expression maintained seed dormancy in dormant ecotypes. Similar conclusions were previously drawn by Chen et al. (2010). By overexpressing AtHD2a, Zhou et al. (2004) showed that this protein could control the development of different plant organs. Indeed, plants that overexpress AtHD2a are characterized by abnormal leaves (i.e. branching or narrow-leaf phenotypes), delayed flowering, aberrant flowers, and aborted seed development. Without overexpression experiments, Kuang et al. (2012) demonstrated that DlHD2 controls senescence in

© 2013 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 1259–1269

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Figure 3. Relative expression of the four type-II histone deacetylases (HD2) genes from Arabidopsis thaliana (a) and the two genes from Oryza sativa (b) in response to various biotic stresses. The search was performed using Genevestigator (Hruz et al. 2008).

longan fruit by potentially interacting with ethyleneresponsive factors. However, for the latter case, the question of how HD2s control these developmental processes remains largely unanswered.

HD2s in the response to abiotic stresses Sridha & Wu (2006) and Luo et al. (2012b) report that the germination rate of various HD2c Arabidopsis mutants (hd2c-1, hd2c-2 and hd2c-3) was strongly affected by ABA or NaCl treatments. Interestingly, using both BiFC (Luo et al. 2012a) and co-immunoprecipitation assays, the authors also demonstrated that this abiotic stress response was achieved through the molecular interaction of HD2c with HDA6 – an RPD3 HDAC. Furthermore, using hda6, hda2-1 and hda6/ hda2-1 double mutants, histone H3 was identified as one of the substrates of the HD2c/HDA6 complex: the acetylation level of H3K9K14 increased whereas the demethylation level of H3K9 decreased (Luo et al. 2012b).

HD2 in the response to biotic stresses Pioneer studies dedicated to HDACs belonging to the RPD3HDA1 family (e.g. Zhou et al. 2005; Wu et al. 2008) indicate that modification of the acetylation levels of nuclear proteins,

particularly of histones, is involved in the control of plant defence responses. More recently, a role for HD2s in plant immunity was also reported. Hence, Bourque et al. (2011) showed that cryptogein, a proteinaceous elicitor secreted by the oomycete Phytophthora cryptogea, induces the rapid phosphorylation of two tobacco HD2 isoforms, namely NtHD2a and NtHD2b, closely related to Arabidopsis AtHDT1 and AtHDT3. Through pharmacological and reverse genetic approaches, NtHD2s were found to act as negative regulators of the hypersensitive response (HR) induced by cryptogein. Indeed, impairment of their expression by RNAi silencing resulted in exacerbated cryptogeininduced cell death in cell suspension lines and in HR-like symptoms in distal leaves. It is interesting to note that in the same silenced cell lines, non-necrotic elicitors, such as oligogalacturonides, did not trigger cell death. This observation suggests that NtHD2a/b do not control the induction of cell death pathways but rather regulate cell death intensity. The mechanisms that underlie the involvement of HD2s in plant defence responses remain speculative at the moment (Dahan et al. 2011a). Notably, the proteins targeted by HD2s during biotic stresses have not been identified yet. Recently, Ding et al. (2012) confirmed and clarified the role of HD2s in the control of plant defence. They first confirmed that overexpression of HDT701, a rice HD2, positively regulates

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Figure 4. Hypothetical model of the mode of action of type-II histone deacetylases (HD2) in the control of the biological response. The different steps of this model are either annotated with numbers when the steps were demonstrated at least for one HD2, or with ‘?’ when the steps are only speculative. (1) Lusser et al. 1997; (2) Ding et al. 2012; (3) Kuang et al. 2012; and (4) Wu et al. 2000; Luo et al. 2012b.

the resistance of rice to Magnaporthe oryzae and Xanthomonas oryzae pv oryzae. Then they showed that HDT701 controls innate immunity by modulating the acetylation of histone H4 in the promoter of defense-related genes in rice such as MAPK6 and WRKY71.

CONCLUSION It is now clear that the modulation of the acetylation levels of both histone and non-histone proteins is of major importance in the control of gene expression. The presence of the HD2 family in plants raises an exciting question as to why evolution provided plants with a third exclusive HDAC family while in other eukaryotic organisms the RPD3-HD1 and SIR2 families are sufficient to carry out the control of gene expression. Interestingly, to our knowledge, HD2s are not involved in the control of basic cellular functions such as cell division or differentiation, but rather in plant-specific processes such as leaf development (Zhou et al. 2004) or response to abiotic stresses (Luo et al. 2012b). Deciphering the signalling pathways that control HD2 biological functions

and their molecular targets (i.e. genes and proteins) represents promising perspectives that will provide a better understanding of how HD2s control plant-specific processes. Figure 4 summarizes current knowledge about this particular field of research. Firstly, it is clear that HD2s are almost exclusively nuclear proteins under resting conditions, suggesting that they are related to the control of gene transcription. As several HD2s contain a NES sequence, nucleocytosolic shuttling could efficiently regulate their activity. The way HD2s are translocated upon perception of specific stimuli remains hypothetical. HD2 phosphorylation by a CK2α activity could also represent a decisive step in the regulation of HD2 functions, although the regulation of CK2α activity in the nucleus is still a matter of debate (Meggio & Pinna 2003; Merson et al. 2006). What are the consequences of HD2 phosphorylation? At least three non-exclusive possibilities can be considered. Firstly, in cryptogein-elicited tobacco, the phosphorylation of NtHD2s is accompanied by an accumulation of hyperacetylated nuclear proteins similarly found in mutants impaired in their expression (Bourque et al. 2011). Thus, phosphorylation most

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HD2 as new nuclear signal transducers likely inhibits HD2 deacetylase activity. Things differ with the other HDAC families in mammalian cells, where CK2α phosphorylation promotes deacetylase activities (e.g. HDAC1; Pflum et al. 2001) or, in some cases, has no effect (e.g. HDAC8; Lee et al. 2004). Secondly, HD2 phosphorylation could lead to a modification of their subcellular localization. This hypothesis was nicely demonstrated in A. thaliana for HDA15 – a member of the RPD3 Class II HDAC – that has both an NLS and a NES and is translocated in response to light (Alinsug et al. 2012). Delocalization of HD2s has never been reported so far, but we have strong evidence that NtHD2s are translocated from the nucleus to the cytosol in response to cryptogein treatment (V. Grandperret and S. Bourque, unpublished results). Once in the cytosoplasm, HD2s could be sequestered by 14-3-3 proteins, as shown with HDA15 in A. thaliana (Alinsug et al. 2012) and/or they could be degraded through the ubiquitin-proteasome pathway (Scognamiglio et al. 2008). Thirdly, phosphorylation could result in changes in the structure of the multimeric complex in which HD2s are thought to be associated, thus modifying their specificity and/or activity. Regardless of the actual molecular consequence(s), HD2 phosphorylation could result in fine in the accumulation of hyperacetylated nuclear proteins that participate in the signal transduction pathways controlled by these proteins. However, HDACs can also regulate cellular processes independently of their deacetylase activity. This was shown by Cabrero et al. (2006) who reported that the expression of a truncated HDAC6 that lacked the deacetylase domain complemented an HDAC6 mutant line and restored lymphocyte chemotaxis. Finally, depending on the stimulus that originally mobilizes HD2s, specific gene expression can be regulated either directly, through for instance the modulation of histone acetylation levels in the vicinity of promoters; or indirectly, through the regulation of the activity of transcriptional regulators such as transcription factors. As shown in this incomplete and mostly speculative model, our knowledge of HD2s is limited. At least two priority points should be clarified: how HD2s are mobilized in the signalling pathways in which they are involved, and what their molecular targets are, i.e. which genes and proteins.

ACKNOWLEDGMENTS We wish to thank our colleagues for helpful discussions. The work in the author’s lab was supported by grants from the Ministère de l’Education Nationale et de la Recherche and from the Conseil Régional de Bourgogne (PARI AGRALE 8). Vincent Grandperret is supported by a PhD fellowship from the Association pour la Recherche sur les Nicotianaées and the Conseil régional de Bourgogne.

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Received 5 September 2013; received in revised form 4 November 2013; accepted for publication 10 November 2013

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