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Arabidopsis HIT4, a regulator involved in heat-triggered reorganization of chromatin and release of transcriptional gene silencing, relocates from chromocenters to the nucleolus in response to heat stress Lian-Chin Wang*, Jia-Rong Wu*, Yi-Ju Hsu and Shaw-Jye Wu Department of Life Sciences, National Central University, 300 Jhong-Da Road, Jhong-Li City, Taoyuan County 32001, Taiwan

Summary Author for correspondence: Shaw-Jye Wu Tel: +886 3 4227971 Email: [email protected] Received: 3 July 2014 Accepted: 22 August 2014

New Phytologist (2015) 205: 544–554 doi: 10.1111/nph.13088

Key words: Arabidopsis thaliana, chromatin reorganization, chromocenter decondensation, heat stress, HEAT-INTOLERANT 4 (HIT4), MORPHEUS’ MOLECULE 1 (MOM1), transcriptional gene silencing.


Arabidopsis HIT4 is known to mediate heat-induced decondensation of chromocenters and

release from transcriptional gene silencing (TGS) with no change in the level of DNA methylation. It is unclear whether HIT4 and MOM1, a well-known DNA methylation-independent transcriptional silencer, have overlapping regulatory functions.
A hit4-1/mom1 double mutant strain was generated. Its nuclear morphology and TGS state were compared with those of wild-type, hit4-1, and mom1 plants. Fluorescent protein tagging was employed to track the fates of HIT4, hit4-1 and MOM1 in vivo under heat stress.
HIT4- and MOM1-mediated TGS were distinguishable. Both HIT4 and MOM1 were localized normally to chromocenters. Under heat stress, HIT4 relocated to the nucleolus, whereas MOM1 dispersed with the chromocenters. hit4-1 was able to relocate to the nucleolus under heat stress, but its relocation was insufficient to trigger the decompaction of chromocenters. The hypersensitivity to heat associated with the impaired reactivation of TGS in hit4-1 was not alleviated by mom1-induced release from TGS.
HIT4 delineates a novel and MOM1-independent TGS regulation pathway. The involvement of a currently unidentified component that links HIT4 relocation and the large-scale reorganization of chromatin, and which is essential for heat tolerance in plants is hypothesized.

Introduction Given that plants are immobile, their growth and development can be hampered seriously if environmental conditions become unfavorable. High temperature is one such adverse condition. It can result in protein denaturation and membrane fluidization and also provoke the accumulation of reactive oxygen species (ROS), which arrest plant growth and even reduce survivability (Wahid et al., 2007). However, plants have evolved many strategies, including morphological and metabolic adjustments, to mitigate the effects of heat injury. Nevertheless, for these strategies to be successful, rapid changes in the global patterns of gene expression are necessary (Larkindale & Vierling, 2008). The heat stress transcription factor (Hsf) signaling system is a well-known regulatory mechanism for reprogramming transcriptional activities in plants under conditions of heat stress (Scharf et al., 2012). In angiosperm species, Hsfs are encoded by large gene families, with numbers of members that range from 19 in castor bean (Ricinus communis) to 56 in wheat (Triticum *These authors contributed equally to this work.

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aestivum) (Scharf et al., 2012; Xue et al., 2014). Hsfs have a modular structure that consist of an N-terminal DNA binding domains, an oligomerization domain and a less conserved C-terminal domain. Plant Hsfs are assigned to three different classes (classes A, B and C) primarily on the basis of peculiarities in the oligomerization domain (Scharf et al., 2012). Although few Hsf members have been characterized in detail, extensive research in the model plant Arabidopsis, which contains 21 Hsf genes, has provided evidence that members of the AtHsfA1 subfamily are expressed constitutively at low levels and serve as master regulators that trigger a transcription cascade by inducing the expression of other transcription factors, including DREB2A, DREB2B, MBF1C, bZIP28 and AtHsfA2 (Liu et al., 2011; Yoshida et al., 2011). AtHsfA2 is the dominant Hsf in thermotolerant plants and is responsible for the expression of genes that encode protein chaperones, such as heat shock proteins, and ROS detoxifying enzymes, such as ascorbate peroxidase 2 (APX2; Schramm et al., 2006; Larkindale & Vierling, 2008). By contrast, the expression of APX1 is mediated by AtHsfA4 (Davletova et al., 2005). The activity of AtHsfA4 is repressed specifically by AtHsfA5 through hetero-oligomerization of the two proteins Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist (Baniwal et al., 2007). Given that AtHsfA4 is induced by H2O2 and plants that overexpress a dominant negative form of AtHsfA4 exhibit hypersensitivity to oxidative stress (Davletova et al., 2005), it is possible that AtHsfA4 functions as a ROS sensor and AtHsfA4 and A5 work together to control ROS homeostasis. Meanwhile, HsfB1 and B2b, which are also heat-inducible but lack a transcription activator motif in their C-terminal domains, act as transcriptional repressors but are necessary for acquired thermotolerance (Ikeda et al., 2011). Although the complexity and functional diversification of the plant Hsf family enables it to form a tightly coordinated network that mediates the activities of heat response genes, recent studies indicate that control at the level of transcriptional gene silencing (TGS) is also involved in the heat-induced modulation of gene expression. In Arabidopsis, certain TGS loci are reactivated under heat stress and silenced again when the stress is removed (Pecinka et al., 2010; Tittel-Elmer et al., 2010). TGS loci are often characterized by DNA hypermethylation at cytidine residues and are packed into heterochromatin (Fransz & de Jong, 2011). In interphase nuclei in Arabidopsis, different heterochromatic regions in a single chromosome can cluster further into a highly compact, subnuclear body stained by 40 , 6-diamidino-2-phenylindole (DAPI) that is known as the chromocenter. However, the heat-induced reactivation of TGS loci occurs without DNA demethylation (Pecinka et al., 2010; Tittel-Elmer et al., 2010). Indeed, DNA demethylation is not obligatory for the release of TGS as illustrated by the mom1 mutation. MORPHEUS’ MOLECULE 1 (MOM1) is a nuclear protein that acts as a transcriptional silencer. Depletion of MOM1 can release a subset of TGS loci without major changes in the pattern of DNA methylation (Amedeo et al., 2000; Tariq et al., 2002). On the other hand, mutation of HEAT-INTOLERANT 4 (HIT4), which encodes a chromocenter localized protein involved in chromatin organization, attenuates the heat-mediated decondensation of chromocenters and release of TGS. These phenomena suggest that HIT4, like MOM1, influences TGS in a DNA methylation independent manner (Wang et al., 2013). However, although mutation of MOM1 promotes release from TGS, mutation of HIT4 inhibits this release. These facts raise a series of important questions: what is the epistatic relationship between hit4 and mom1? Do HIT4 and MOM1 act antagonistically in the DNA methylation-independent regulation of TGS? If so, how do they impose their opposing effects on the heat-induced reactivation of TGS and heat tolerance in plants? If not, what are their distinct regulatory functions? Furthermore, and equally important, how does HIT4 destabilize the structural integrity of chromocenters under heat stress? To address these fundamental and critical questions, we constructed a hit4-1/mom1 double mutant strain and analyzed its function in TGS under non-stressed and heat-stressed conditions. We also generated stable transgenic plants that express fluorescent-protein-tagged HIT4 and MOM1 to track the fates of HIT4 and MOM1 in vivo under heat stress. The results indicated that HIT4 delineates a novel TGS regulatory pathway that functions independently of MOM1, and revealed the Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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potential regulatory function of HIT4 in the heat-triggered reorganization of chromatin.

Materials and Methods Plant materials, growth conditions and heat treatment procedures Wild-type Arabidopsis thaliana (L.) Heynh seeds in the Columbia-0 background were purchased from Lehle Seeds (Round Rock, TX, USA). The mutant lines of hit4-1 and mom1 (SAIL_610_G01) that were used in the present study were as described previously (Wang et al., 2013). The hit4-1/mom1 double mutant was generated by crossing hit4-1 plants to mom1. Homozygous hit4-1/mom1 progeny were determined by PCRbased genotyping. The primers hit4-1-NdeI-F and hit4-1-NdeI-R were used to genotype hit4-1 (Supporting Information Table S1). These primers amplified a 151-bp fragment. The amplicon derived from hit4-1 but not that from the wild-type, was digested by the restriction endonuclease NdeI to generate 128- and 23-bp fragments. In addition, three primers were used in combination to genotype mom1. Whereas MOM1-503F and MOM1-1197R amplified a 695-bp wild-type sequence, MOM1-503F and LB3 amplified a 499-bp sequence from the junction of the T-DNA insert and plant DNA in the mutant (Table S1). Detailed procedures for medium preparation, seed sterilization, stratification, and germination were as described by Lee et al. (2006) and Wang et al. (2011). Plants grown in medium were cultured in a growth chamber set at 23°C with continuous light. Detailed procedures for heat-stress treatment of the medium-grown plants were as described by Wu et al. (2010). For the purposes of crossing mutant lines, Agrobacterium-mediated genetic transformation, and seed propagation, plants were grown in soil at 23°C under long-day conditions with a 16 h : 8 h, light : dark photoperiod. The light intensity for both medium- and soil-grown plants was 100 lmol m2 s1. Gene expression analysis by quantitative real-time PCR To analyze heat-triggered release from TGS, 14-d-old plants grown in medium were transferred to a growth chamber set at 37°C and incubated for 0, 3, 6, 12, 24 or 36 h. At the end of the exposure to heat, c. 50 seedlings were collected for total RNA extraction. The total RNA was prepared using the GeneMark Plant Total RNA Miniprep Purification Kit (Hopegen Biotechnology, Taichung, Taiwan) in accordance with the manufacturer’s protocol. Quantitative real-time PCR (qPCR) was performed using the KAPA SYBR FAST One-Step qRT-PCR Kit (KAPA Biosystems Inc., Woburn, MA, USA) in accordance with the manufacturer’s instructions under the following conditions: 42°C for 5 min (cDNA synthesis), 95°C for 5 min (to inactivate reverse transcription), and 40 cycles of 95°C for 3 s (for denaturation) and 60°C for 1 min (for annealing and extension). The regions amplified and the primers used were as follows: 180-bp repeat with the primers 180 bp(all)-F and 180 bp(all)-R; the New Phytologist (2015) 205: 544–554 www.newphytologist.com

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TRANSCRIPIONALLY SILENT INFORMATION (TSI) repeat with the primers TSI-qF and TSI-qR; ONSEN with the primers ONSEN-qF and ONSEN-qR; MULE-F19G14 with the primers MULE-qF and MULE-qR (Table S1). UBC28 was also amplified with the primers UBC28-qF and UBC28-qR as an internal control for quantitative normalization (Table S1). All qPCR reactions were performed in triplet replicates of independent biological experiments and analyzed with the BioRad iCycler IQ5 Real-Time PCR Detection System (Life Science Research, Hercules, CA, USA). Transgenic plants to track protein localization To analyze the localization of HIT4 and hit4-1, the promoter region of HIT4 was amplified by PCR from Col-0 wild-type genomic DNA with the primers pHIT4-EcoRI-F and pHIT4PstI-R (Table S1). The resultant amplicon (942 bp) was digested with EcoRI and PstI and cloned into the pBlueScript II (pBSII) vector (Agilent Technologies Inc., Santa Clara, CA, USA). The HIT4 promoter fragment in pBSII was then released by digestion with EcoRI and SacI and subcloned into the plant transformation vector pCAMBIA1300 (CAMBIA, Canberra, ACT, Australia) to generate pCAMBIA1300-pHIT4. The HIT4 and hit4-1 cDNA fragments were amplified using the primers HIT4-KpnI-F and HIT4-BamHI-R (Table S1) and RNA extracted from wild-type and hit4-1 plants, respectively. The amplified fragments were digested with KpnI and BamHI and cloned into pCHF3-35S -GFP (Jarvis et al., 1998) to generate pCHF3-35S-HIT4-GFP and pCHF3-35S-hit4-1-GFP. The coding regions of HIT4-GFP and hit4-1-GFP fusions constructs were excised by digestion with SacI and HindIII, and then subcloned into pCAMBIA1300pHIT4 to generate pCAMBIA1300-pHIT4-HIT4-GFP and pCAMBIA1300-pHIT4-hit4-1-GFP, respectively. To analyze the localization of MOM1, the coding region of MOM1 was amplified with the primers MOM1-KpnI-F and MOM1-BamHI-R (Table S1). The resultant amplicon was cloned first into the yTA vector (Yeastern Biotech, Taipei, Taiwan), and then subcloned into pCHF3-35S-GFP using the KpnI and BamHI restriction sites to generate pCHF3-35S-MOM1GFP. The vectors that contained the desired fusion constructs were introduced individually into wild-type and mutant (hit4-1) Arabidopsis plants by the Agrobacterium-mediated floral dipping method (Clough & Bent, 1998). Positive transformants were selected on half-strength Murashige–Skoog (MS) medium (Murashige & Skoog, 1962) that contained 50 lg ml1 kanamycin or hygromycin. The antibiotic-resistant T1 seedlings were transferred to soil and grown to maturity. Homozygous T2 plants were selected by growth of their T3 generations on medium that contained kanamycin. T3 seeds derived from homozygous T2 plants were used for subsequent analysis of protein localization.

from RNA extracted from wild-type plants by PCR using the primers FIB1-BamHI-F and FIB1-EcoRI-R (Table S1). The amplified fragment was digested with BamHI and EcoRI and cloned into the vector pLOLA-35S-mCherry (Wang et al., 2011) to generate pLOLA-35S-mCherry-FIB1. For transient co-expression of fluorescent-protein-tagged HIT4 and MOM1 to analyze their relocation in response to exposure to high temperature, HIT4 cDNA was amplified by PCR using RNA extracted from wild-type plants as the template and the primers HIT4-BamHI-F and HIT4-PstI-R (Table S1). The resultant amplicon was digested with BamHI and PstI and cloned into the vector pLOLA-35S-mCherry to generate pLOLA-35SmCherry-HIT4. To increase the efficiency of protoplast transformation, the 35S-GFP coding region was excised from the relatively large binary vector pCHF3-35S-GFP by digestion with EcoRI and HindIII and subcloned into pUC18 to generate the pUC18-35SGFP construct. In addition, the MOM1 cDNA was amplified by PCR with the primers MOM1-KpnI-F and MOM1-BamHI-R (Table S1), digested with KpnI/BamHI, and then inserted into pUC18-35S-GFP to generate pUC18-35s-MOM1-GFP. Transient gene expression for analysis of protein localization Fully expanded rosette leaves of 4-wk-old Arabidopsis plants were used to prepare protoplasts. The lower epidermis of the leaves was peeled away and floated on an enzyme solution that contained 0.4 M mannitol, 20 mM KCl, 10 mM CaCl2, 1% (w/v) cellulase R10, 0.25% (w/v) macerozyme R10, 0.1% (w/v) bovine serum albumin (BSA), 20 mM 2-(N-morpholino)ethanesulfonic acid (MES), and 5 mM b-mercaptoethanol, pH 5.7 for 45 min with gentle shaking at 40 rpm. The released protoplasts were collected by centrifugation at 100 g for 1 min in a 50 ml centrifuge tube. The pelleted protoplasts were resuspended in 10 ml W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM glucose, 2 mM MES, pH 5.7) and then centrifuged again. The collected protoplasts were resuspended in 10 ml of W5 solution and incubated on ice for 30 min, after which the protoplasts were centrifuged and resuspended in MMg solution (0.4 M mannitol, 15 mM MgCl2, 4 mM MES, pH 5.7) at 2 9 105 cells ml1. Polyethylene glycol (PEG)-mediated transformation was performed by gently mixing 20 ll of plasmid DNA (10 lg), 220 ll of 40% PEG (in 0.2 M mannitol and 0.1 M CaCl2) and 200 ll of protoplasts (4 9 104 cells). The mixture was incubated for 10 min at 22°C, and then 1 ml of W5 solution was added to the mixture, which was centrifuged for 1 min at 100 g. The collected protoplasts were washed again with 1 ml of W5 solution, resuspended in 1 ml of W5, and incubated at 22°C for 12–16 h. Expression of the fluorescent fusion proteins was observed and photographed under an Olympus IX71 fluorescence microscope (Center Valley, PA, USA).

Constructs for protoplast transformation

DAPI staining of root cells

For the nucleolus localization assay, the cDNA fragment for Arabidopsis FIBRILLARIN1 (FIB1, At5g52470) was amplified

Ten-day-old seedlings that had been subjected to heat-stressed or non-stressed conditions were incubated in 1 lg ml1 DAPI for

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New Phytologist 30 min at room temperature. The DAPI signals were visualized and photographed by fluorescence microscopy using an Olympus IX71 fluorescent microscope.

Results Regulation of TGS mediated by HIT4 or MOM1 is distinguishable As a starting point, we measured the transcript levels of several endogenous TGS markers that are known to be reactivated by high temperatures and compared WT, hit4-1, mom1 and the hit4-1/mom1 seedlings. The markers included the centromeric 180-bp repeat, the pericentromeric TSI repeat, the multicopy ONSEN retrotransposon, and the single copy MULE-F19G14 locus (Steimer et al., 2000; Ito et al., 2011; Matsunaga et al., 2012). The heat-induced reactivation of these TGS loci was found previously to be attenuated in the hit4-1 mutant (Wang et al., 2013). With the exception of ONSEN, these markers are transcriptionally activated in the mom1 background under nonstressed growth conditions (Pecinka et al., 2010; Tittel-Elmer et al., 2010). In the hit4-1/mom1 mutant plants, the normally silent 180-bp repeat, TSI, and MULE-F19G14 were activated either before or after prolonged exposure to 37°C for 36 h to extents that were similar to those observed in mom1. By contrast, although transcription of ONSEN was activated in hit4-1/mom1 after heat treatment, the amount of transcript was considerably less than that observed in the WT and mom1, and identical to that detected in hit4-1 mutant plants (Fig. 1). These results demonstrated that there are two distinguishable control mechanisms for TGS that involve either HIT4 or MOM1, respectively. These results imply also that, whereas some TGS loci are regulated by both HIT4 and MOM1 (180-bp, TSI, and MULE), some (e.g. ONSEN in the present study) are controlled specifically by HIT4.

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Heat-induced decondensation of chromocenters occurs in the mom1 single mutant but not in the hit4-1/mom1 double mutant In addition to the constraint of heat-induced release of TGS, mutation of HIT4 also results in restriction of the heat-induced decondensation of chromocenters (Wang et al., 2013). Given that HIT4 and MOM1 are involved in distinct regulatory pathways for TGS, as indicated above, it was considered likely that the phenomenon of maintaining compact chromocenters under heat stress would co-segregate with hit4-1 and appear in hit4-1/ mom1 double mutant plants. To verify this, roots of 10-d-old seedlings were stained with the fluorescent dye DAPI and the nuclei of cells in the zones of elongation and maturation were monitored for heat-mediated changes in chromocenter morphology. Before exposure to heat stress, compact chromocenters were clearly visible in most nuclei (> 70%) of hit4-1/mom1, which was similar to the levels observed in the WT, hit4-1, and mom1 (Fig. 2). After exposure to heat for 30 h at 37°C or heat shock for 30 min at 44°C, heat-triggered dispersion of chromocenters was obvious in the WT and mom1, as judged by diffuse nucleoplasmic labeling with DAPI. However, most nuclei (> 70%) of hit4-1/mom1 maintained discernible DAPI fluorescence in a subnuclear domain as observed for hit4-1 (Fig. 2). These results indicated that HIT4 but not MOM1 is associated with heatinduced decompaction of the chromocenters. HIT4 relocates from chromocenters to the nucleolus before heat-induced decondensation of chromocenters occurs Given that heat-induced decondensation of chromocenters is restricted in hit4-1 but not in mom1, and previous transient expression analysis using green fluorescent protein (GFP)-tagged HIT4 protein in Arabidopsis mesophyll protoplasts revealed that HIT4 is localized to chromocenters at 23°C (Wang et al., 2013),

Fig. 1 HIT4 is involved in a control mechanism for transcriptional gene silencing (TGS) that is distinguishable from that of MOM1. qRT-PCR analysis was performed to quantify the levels of the 180-bp repeat, MULE, TSI, and ONSEN transcripts, whose expression is normally under TGS control and is reactivated by prolonged heat exposure. RNA for analysis was extracted from Arabidopsis thaliana wild-type (WT), hit4-1, mom1, and hit4-1/mom1 seedlings that had been incubated for various time periods at 37°C. Relative expression levels were calculated and normalized relative to UBC28 transcript. Values are the mean  SD of three biological replicates (i.e. three independently isolated RNA samples at each time point, n = 3). Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Fig. 2 Chromocenter decondensation induced by heat stress occurs in the mom1 single mutant but not in the hit4-1/mom1 double mutant. (a) Phenotypes of representative DAPI-stained root interphase nuclei in Arabidopsis thaliana wild-type, hit4-1, mom1, and hit4-1/mom1 seedlings before and after prolonged heat stress for 30 h at 37°C or heat shock for 30 min at 44°C. Chromocenter decondensation was assessed by the appearance of diffuse labeling of the nucleoplasm with DAPI. WT, wild-type; RT, room temperature (23°C) without heat stress; bar, 5 lm. (b) Quantification of nuclei with condensed chromocenters in the roots of WT, hit4-1, mom1, and hit4-1/mom1 seedlings before and after heat stress at 37°C for 30 h or heat shock at 44°C for 30 min. The total number of nuclei and the number of nuclei with condensed chromocenters were counted from the zone of elongation toward the zone of maturation, and are expressed as the percentage of nuclei with condensed chromocenters (CC). Fifty nuclei were counted from each seedling root; values are mean  SD of five seedlings from each treatment. WT, wild-type; RT, room temperature (23°C) without heat treatment. New Phytologist (2015) 205: 544–554 www.newphytologist.com

it was reasonable to assume that tracking the localization of HIT4 in situ under high temperature might help elucidate the role of HIT4 in chromocenter organization and TGS regulation. In this regard, we generated a stable line of transgenic Arabidopsis by transforming the HIT4 promoter-driven HIT4-GFP fusion construct into the hit4-1 background. The transgenic plants were first tested for their ability to complement the heat-intolerant phenotype of hit4-1. As shown in Fig. 3(a), the transgenic hit4-1 plants that expressed HIT4-GFP tolerated both sustained high temperature and sudden heat-shock treatments, in a similar manner to WT seedlings, which indicated that the GFP-tagged HIT4 was molecularly functional. Ten-day-old transgenic seedlings were subjected to heat treatment at 37°C and 44°C for various durations and the fluorescent signals in root cell nuclei were observed under a fluorescent microscope. Initially, GFP signals that were localized at chromocenters disappeared and aggregated into a single subnuclear domain in each nucleus within 1 h at 37°C or 5 min at 44°C (Fig. 3b). By contrast, chromocenters themselves remained conspicuous without noticeable morphological change at these time points, and clear decompaction of chromocenters was seen only after the seedlings had been heat stressed for 30 h at 37°C or for 30 min at 44°C (Figs 2, 4b). On the basis of its number and morphology, the subnuclear compartment to which HIT4-GFP was localized upon heat treatment corresponded most probably to the nucleolus. This hypothesis was tested by examining Arabidopsis protoplasts that co-expressed HIT4-GFP and a nucleolar marker protein FIB1 (Reichow et al., 2007) tagged with mCherry. The GFP signal co-localized with mCherry after the protoplasts had been incubated for 1 h at 37°C, which indicated that HIT4 indeed relocated to the nucleolus in response to heat treatment (Fig. 3c). To investigate the nature of the heat-induced relocation of HIT4 further, HIT4-GFP transgenic seedlings that had been subjected to heat stress at 37°C for 1 h or 44°C for 5 min were transferred to 23°C and their fluorescent signals were monitored continuously. The nucleolus-localized GFP signals were observed to return to chromocenters after recovery for 1 d at 23°C (Fig. 3b). Collectively, these results suggested that the heatinduced HIT4-mediated decondensation of chromocenters involves both temporal and spatial factors, and demonstrated that relocation of HIT4 in response to high temperature is a reversible process. Relocalization of HIT4 alone is insufficient to promote heatinduced decondensation of chromocenters Given that hit4-1 is a missense mutation in which the codon for Ser 227 is changed to Tyr (Wang et al., 2013), it was envisaged that the hit4-1 phenotypes result from the altered function of the HIT4 protein. The relocation of HIT4 from chromocenters to the nucleolus in response to heat stress and the observation that the relocation occurred before heat-induced decompaction of chromocenters prompted us to speculate that the function of HIT4 is to maintain the condensed structure of chromocenters and that the hit4-1 mutant protein had an impaired ability to leave chromocenters under high temperature, which thus Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Fig. 3 Chromocenter-localized HIT4 migrates to the nucleolus before heat-induced decondensation of chromocenters occurs. (a) Transformation of the hit4-1 mutant with a HIT4 promoter-driven HIT4-GFP fusion construct restored its ability to tolerate heat to that of the wild-type, which demonstrated that the fusion protein was molecularly functional. To induce prolonged heat stress, 10-d-old Arabidopsis thaliana plants grown in medium were transferred to 37°C and incubated for 4 d. After heat treatment, the plants were allowed to recover at 23°C for 3 d before the image was taken. For sudden heat shock, 10-d-old seedlings were exposed to 44°C for 30 min followed by recovery at 23°C for 7 d before the image was taken. Growth of h4H4G seedlings without heat stress treatment is shown in Supporting Information Fig. S1. WT, untransformed wild-type; h4H4G, hit4-1 transformed with pHIT4HIT4-GFP. (b) Representative fluorescent images of nuclei from root cells of pHIT4-HIT4-GFP transgenic plants subjected to different heat stress regimes as shown schematically to the left of the photographs. The time points at which the images were taken are indicated by arrow heads. The DAPI signal in the merged panels is pseudo-colored in red for better distinction of the GFP signal in the chromocenter or nucleolus. Bar, 5 lm. (c) Protoplasts from Arabidopsis leaf tissue were co-transformed with expression vectors for HIT4-GFP and the nucleolus marker mCherry-FIB1. At 16 h after transformation by polyethylene glycol-mediated transfection, the cells were incubated for an additional 1 h at 37°C before the images were taken. Bar, 10 lm.

stress for 1 h at 37°C or 5 min at 44°C, the chromocenterlocalized green fluorescence was replaced by nucleolus-localized green fluorescence (Fig. 4b). These changes in the localization of the hit4-1-GFP signals persisted after the seedlings were heat stressed for 30 h at 37°C or for 30 min at 44°C. At these time points, the chromocenters in the transgenic plants remained condensed and were discernible as DAPI-stained spots, whereas those in the WT were dispersed (Fig. 4b). Therefore, the missense hit4-1 mutation did not affect the ability of its protein product to relocate in response to heat stress, and correct relocalization of HIT4 alone was insufficient to promote heat-induced decondensation of chromocenters. (c)

restricted heat-triggered decondensation of chromocenters. To examine this hypothesis, we generated a stable line of transgenic Arabidopsis by transforming the hit4-1-GFP fusion construct driven by the HIT4 promoter into hit4-1 plants. The resultant transgenic plants were tested first for their heat sensitivity. The plants were intolerant to both sustained heat stress at 37°C for 4 d and sudden heat shock at 44°C for 30 min, as is the case for hit4-1 plants as expected (Figs 4a, S1). Green fluorescence in root cell nuclei of 10-d-old hit4-1-GFP transgenic hit4-1 plants was monitored under a fluorescent microscope. Initially, green fluorescence was observed in the chromocenters (Fig. 4b). After heat Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

Chromocenter-localized MOM1 does not relocate to the nucleolus in response to heat stress The MOM1 protein contains nuclear localization signal (NLS) sequences. Immunodetection of HA-epitope-tagged MOM1 expressed transiently in tobacco (Nicotiana tabacum) BY-2 cells demonstrated that MOM1 is localized to the nucleus (Amedeo et al., 2000). However, in contrast to Arabidopsis, which has a small genome (130 Mbp, n = 5), N. tabacum is an amphiploid species with a very large genome (n = 24, 1C = 5.2 pg; Leitch et al., 2008). Consequently, the majority of the tobacco nucleus consists of heterochromatin, which does not display chromocenters (Libault et al., 2005). To investigate the subnuclear localization of MOM1 in Arabidopsis in detail, and to clarify the relationship between HIT4 and MOM1 in the control of TGS and heat-induced reorganization of chromocenters, we stably introduced a MOM1-GFP fusion construct driven by the 35S promoter into wild-type Arabidopsis plants. These transgenic plants showed fluorescent foci in their nuclei, which were identified easily by the observation of root cells under a fluorescent New Phytologist (2015) 205: 544–554 www.newphytologist.com

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Continuous monitoring revealed that MOM1 remained localized at chromocenters after the transgenic seedlings had been heat stressed for 1 h at 37°C or 5 min at 44°C (Fig. 5a), under which conditions HIT4 had relocated to the nucleolus. After heat stress for 30 h at 37°C or for 30 min at 44°C, during which heatinduced decondensation of chromocenters occurred, the MOM1-GFP fluorescent foci dispersed in a pattern identical to that of the DAPI-stained foci. To corroborate further that the subnuclear localization of HIT4 and MOM1 in response to high temperature differed, protoplasts derived from Arabidopsis cell suspensions were transformed for transient co-expression of mCherry-HIT4 with MOM1-GFP. Before heat treatment, fluorescently tagged HIT4 and MOM1 were colocalized to chromocenters. After heat stress for 1 h at 37°C or 5 min at 44°C, mCherry fluorescence dissipated from chromocenters and appeared in the nucleolus, whereas GFP fluorescence remained at the chromocenters (Fig. 5b). Thus, HIT4 and MOM1 indeed become separated spatially under heat stress. Physiological effects derived from the hit4-1 mutation are not compensated by the mom1 mutation

Fig. 4 The hit4-1 mutant protein relocates from chromocenters to the nucleolus in response to heat stress, but does not mediate heat-triggered chromocenter decondensation. (a) Transformation of the hit4-1 mutant with a HIT4 promoter-driven hit4-1-GFP fusion construct did not restore thermosensitivity to the level of the wild-type, which demonstrated that the fusion protein is impaired functionally as is the hit4-1 protein. Plants grown in medium were transferred either to 37°C for 4 d or to 44°C for 30 min and were allowed to recover at 23°C for 3 or 7 d, respectively, before photography. Growth of h4h4G seedlings without heat stress treatment is shown in Fig. S1. WT, untransformed wild-type; h4h4G, hit41 transformed with pHIT4-hit4-1-GFP. (b) Representative fluorescent images of nuclei from root cells of transgenic plants expressing either the HIT4-GFP or hit4-1-GFP fusion construct. Ten-day-old transgenic seedlings were subjected to different heat stress regimes as shown schematically to the left of the photographs. The time points at which the pictures were taken are indicated by arrow heads. DAPI signal in the merged panels is pseudo-colored in red for better distinction of the GFP signal in the chromocenter or nucleolus. Bar, 5 lm.

microscope. When counterstained with DAPI, clear colocalization of MOM1 foci with chromocenters was indicated by the perfect overlay of the GFP and DAPI fluorescent signals (Fig. 5a). New Phytologist (2015) 205: 544–554 www.newphytologist.com

We then compared the hit4-1 and mom1 single mutants and hit4-1/mom1 double mutant at the physiological level by testing their survivability after heat stress treatment. To induce prolonged high temperature stress, 10-d-old seedlings grown in medium were transferred to an incubator at 37°C. After treatment for 4 d, mom1 seedlings remained green in color, similar to WT seedlings, whereas hit4-1 and hit4-1/mom1 seedlings were totally bleached (Figs 6, S1). For sudden heat-shock treatment, 10-d-old seedlings grown in medium were exposed to 44°C for 30 min, and then returned to 23°C for further observation. After recovery for 7 d at 23°C, the wild-type and mom1 seedlings remained alive and exhibited noticeable growth as judged by the emergence and expansion of true leaves, whereas the leaves of hit4-1 and hit4-1/mom1 seedlings were bleached completely and no signs of growth were observed (Fig. 6). These results demonstrated that the mutant of HIT4 and MOM1 showed differential capabilities for thermotolerance, and implied that the physiological effects that resulted from restriction of the heat-triggerred reactivation of TGS in hit4-1 are not compensated by mom1promoted release from TGS.

Discussion The Arabidopsis hit4-1 mutant line was isolated originally through forward genetic screening on the basis of being defective in basal thermotolerance. Subsequent gene mapping revealed that HIT4 encodes a previously unstudied protein and that the hit4-1 mutation is a missense mutation. Further analysis showed that HIT4 is localized to heterochromatic chromocenters in the interphase cell nuclei of Arabidopsis, and the hit4-1 mutation restricts heat-triggered decondensation of chromocenters and release from TGS (Wang et al., 2013). These findings not only provide strong evidence that links the reorganization of chromatin and reactivation of TGS with heat tolerance in plants, but also raised several Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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(a)

Fig. 6 The heat stress phenotypes of WT, hit4-1, mom1, and hit4-1/ mom1 seedlings. Medium-grown plants of the Arabidopsis thaliana WT, hit4-1, mom1, and hit4-1/mom1 mutants were subjected to prolonged heat stress for 4 d at 37°C or sudden heat shock for 30 min at 44°C. Heat stressed and heat shocked plants were allowed to recover at 23°C for 3 and 7 d, respectively, before photography. Growth of mom1 and hit4-1/ mom1 seedlings without heat stress treatment is shown in Fig. S1. WT, wild-type; h4m1, hit4-1/mom1.

(b)

Fig. 5 MOM1 is localized to chromocenters, did not migrate to nucleolus, but dispersed together with the chromocenters in response to heat stress. (a) Representative fluorescent images of nuclei from the root cells of transgenic plants expressing a 35S promoter-driven MOM1-GFP fusion construct. Tenday-old transgenic Arabidopsis thaliana seedlings were subjected to different heat stress regimes as shown schematically to the left of the photographs. The time points at which the pictures were taken are indicated by arrow heads. Bar, 5 lm. (b) Protoplasts from Arabidopsis leaf tissue were cotransformed with expression vectors for the mCherry-HIT4 and MOM1-GFP fusion construct. At 16 h after transformation by polyethylene glycolmediated transfection, cells were incubated either for an additional 1 h at 37°C or 5 min at 44°C before the images were taken. Bar, 5 lm.

important questions regarding how these responses are regulated. First, given that heat-induced release from TGS does not involve changes in the level of DNA methylation and is not a side effect Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

of DNA damage and/or senescence (Pecinka et al., 2010), heattriggered reactivation of TGS mediated by HIT4 should be DNA demethylation-independent. Among the many other proteins that are involved in maintenance of TGS in a DNA methylation-independent manner, all function in DNA repair except for MOM1 (Takeda et al., 2004; Elmayan et al., 2005; Kapoor et al., 2005; Shaked et al., 2006; Xia et al., 2006; Wang et al., 2007). Moreover, both HIT4 and MOM1 are plant-specific pro
teins (Caikovski et al., 2008; Wang et al., 2013). Thus, it was reasonable to postulate that HIT4 and MOM1 participate in the same TGS control pathway, possibly in an antagonistic manner, because a mutation in HIT4 restricts TGS release whereas a mutation in MOM1 promotes TGS release. However, the TGS loci that are released in mom1 (180-bp repeat, TSI, and MULEF19G14) remained released in the hit4-1/mom1 double mutant at a normal growing temperature (23°C). In addition, the TGS locus that is reactivated specifically by high temperatures (ONSEN) was still restricted in the hit4-1/mom1 double mutant after heat exposure (for 36 h at 37°C), which indicated that at least two distinct DNA methylation-independent TGS regulatory pathways exist, which involve HIT4 and MOM1, respectively. Given that HIT4 is localized normally to chromocenters, and chromocenters decondense under high temperature, it is important to know the fate of HIT4 in response to heat stress to elucidate its regulatory function. Tracking the fluorescent signal of GFP-tagged HIT4 showed that the protein relocated from chromocenters to the nucleolus after heat treatment. This relocation occurred much earlier than the initiation of chromocenter decondensation, and the nucleolus-localized HIT4 returned to chromocenters once cells were relieved from heat stress (Fig. 3). These results indicate that the regulatory function of HIT4 involves both temporal and spatial dimensions. Given that hit4-1 is a missense mutation, it is tempting to speculate that the inability of chromocenters in hit4-1 nuclei to decondense in response to heat stress might result from the impaired ability of the hit4-1 protein to move correctly from chromocenters to the nucleolus. If this is New Phytologist (2015) 205: 544–554 www.newphytologist.com

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the case, GFP-tagged hit4-1 would probably remain at chromocenters under heat stress. However, our observations disproved such speculation because the distribution of hit4-1-GFP fluorescence in hit4-1 nuclei showed the same dynamic pattern as HIT4-GFP in WT nuclei. Namely, hit4-1 relocated to the nucleolus as rapidly as HIT4 after the initiation of heat treatment (within 1 h under heat stress at 37°C or after heat shock for 5 min at 44°C). Moreover, when the duration of heat treatment reached the times at which the chromocenters in the nuclei of WT cells dispersed (30 h at 37°C or 30 min at 44°C), the hit4-1-GFP signal was located continuously at the nucleolus and the DAPI-stained spots that represented condensed chromocenters remained apparent in the nuclei of hit4-1 cells. These results not only demonstrated that the hit4-1 mutation does not impair the ability of hit4-1 protein to relocate from chromocenters to the nucleolus, but also indicated that the temporal and spatial translocations of HIT4 alone are not sufficient to account for the heat-induced decondensation of chromocenters. What, then, does HIT4 translocation contribute to chromocenter decondensation? The finding that hit4-1 can move from chromocenters to the nucleolus and that chromocenters remain condensed in hit4-1 nuclei under heat stress negate the possibilities that HIT4 is a lock that clasps heterochromatic regions together, or that HIT4 is an obstacle to the key that unlocks the compact structure of chromocenters. Instead, HIT4 might be a mediator whose function is to interpret environmental signal and then remove the true obstacle and/or other chromocentermaintaining molecules. In such a scenario, the nucleolus would serve as a temporary storage for HIT4-carrying molecules. It is unlikely that MOM1 is one of the molecules removed by HIT4, because MOM1 remains associated with chromocenters after HIT4 has moved to the nucleolus, which correlates with the aforementioned notion that HIT4 and MOM1 regulate TGS through different pathways. In the future, employing a pulldown approach to identify HIT4-interacting partner(s) and screening for mutants that restrict heat-induced relocation of HIT4 from the chromocenters to nucleolus by using the available HIT4-GFP transgenic line should clarify this issue. ONSEN is a Ty1/copia-type long terminal repeat retrotransposon that exists in multiple copies in the Arabidopsis genome and is normally under TGS control. Its expression is activated specifically by heat stress and it remains inactive in mutants that are defective in DNA methylation-dependent or -independent TGS regulatory pathways (Pecinka et al., 2010; Tittel-Elmer et al., 2010). Recently, it was reported that the ONSEN promoter does not contain two of the three sequences that undergo cytosine methylation: GC and CHG, where H stands for A, T or C. In addition, ONSEN remains silent in the ddm1/drm2/ cmt3 triple mutation background, which lacks RdDM-mediated methylation at CHH sites, the third sequence for cytosine methylation (Cavrak et al., 2014). These findings indicate that activation of ONSEN is independent of DNA methylation. Furthermore, the long terminal repeat of all ONSEN copies contains heat stress element with the consensus sequence nTTCnnGAAn that is recognized by Hsfs, and heat activation New Phytologist (2015) 205: 544–554 www.newphytologist.com

of ONSEN is inhibited or severely reduced in the hsfa1 and hsfa2 mutants, respectively, which explains the heat-inducible nature of ONSEN. However, the heat reactivation of ONSEN is attenuated, and the expression of heat shock protein genes upon heat stress is not affected, in hit4-1 (Wang et al., 2013). This indicates that HIT4 is involved in a TGS regulatory mechanism that controls an additional factor that either keeps ONSEN in silent state or releases it from the silent state, and this factor is associated with the structural stability of chromocenters. From this viewpoint, it is plausible to postulate that HIT4 protects against thermal stresses by reconfiguring chromatin to enable transcriptional activators that include HSFs to access and act at the promoter regions of genes involved in heat tolerance in plants. Furthermore, although all eukaryotic organisms contain heterochromatin, not all species display chromocenters. This fact has led to the suggestion that the appearance of chromocenters is a reflection of genome size and the organization of heterochromatic and euchromatic domains along the linear chromosome (Fransz, 2009). Large-scale decondensation of chromatin is not restricted to heterochomatic sequences, but also affects euchromatic domains and is exemplified during the floral transition (Tessadori et al., 2007b). Therefore, HIT4dependent heat-responsive genes might not necessarily reside within chromocenters. This postulation can also explain the observation that, despite the involvement of both HIT4 and MOM1 in DNA methylation-independent control of TGS, they operate in different manners and scopes; HIT4 regulates the global reorganization of chromatin, whereas MOM1 targets selected loci. In addition to heat stress, chromocenter decondensation in Arabidopsis is also induced by low light intensity and occurs during germination, protoplastization and flowering (Mathieu et al., 2003; Tessadori et al., 2007a,b; Van Zanten et al., 2012). Although it is intuitive and reasonable to suppose that the decondensation of chromocenters is required for reprogramming of gene expression to enable adaptation to environmental changes and developmental phase transition, the mechanism by which chromocenter decondensation is initiated, the genetic components involved, and the means of regulation of downstream genes remain unknown. Thus, the present results provide important insights towards answering these questions. In terms of the response to high temperature, at least an upstream signal that directs HIT4 relocation, a possible molecule or molecules transported away from chromocenters by HIT4, and a subset of genes whose activities rely on HIT4mediated chromatin reorganization and are required for basal thermotolerance in plants need to be identified. The hit4-1 mutant is undoubtedly an invaluable tool to assist in answering the above questions.

Acknowledgements This work was supported by the National Science Council, Taiwan (general research project grants 102-2311-B-008-001MY3 to S-J.W. and postdoctoral fellowship 102-2811-B-008001 to L-C.W.). Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Supporting Information Additional supporting information may be found in the online version of this article.

Fig. S1 Phenotypes of wild-type, hit4-1, mom1, h4H4G, h4h4G, and h4m1 seedlings before heat stress treatment. Table S1 Primer list Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

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