-Hui Shen

Huijia Kang

Chi Zhang

Zengxuan An

Wen-Hui Shen

Yan Zhu
email: zhu_yan@fudan.edu.cn

Keywords: Arabidopsis, ARP5, ARP6, chromatin-remodeling factors, epigenetic regulation, genome stability, INO80-C, SWR1-C

AtINO80 and AtARP5 physically interact and play common as well as distinct roles in regulating plant

growth and development

Introduction

SWI2/SNF2-type chromatin-remodeling factors are macromolecular machines that generally function as crucial effectors in diverse epigenetic regulatory processes.

These factors, which use energy from ATP hydrolysis to slide, eject or restructure nucleosomes, actively modulate the dynamic chromatin structure in response to endogenous developmental programming or external environmental signals (reviewed in [START_REF] Clapier | The biology of chromatin remodeling complexes[END_REF]. A common structure that directly uses ATP fuel, the Snf2 domain, is shared by these SWI2/SNF2-type chromatin remodelers [START_REF] Narlikar | Mechanisms and functions of ATP-dependent chromatin-remodeling enzymes[END_REF]. Based on characteristic domains flanking the Snf2 domain, remodelers from diverse organisms can be briefly classified into Swi/Snf-, Iswi-, Chd-and Ino80-families. While some chromatin-remodeling factors are considered to act alone, many exist as scaffolds interacting with other subunits of varying numbers, which collectively constitute distinct structural and functional modules (reviewed in [START_REF] Clapier | The biology of chromatin remodeling complexes[END_REF].

The Ino80-family comprises two important, related members: INO80 and SWR1.

The name INO80 stems from its initial identification as a transcriptional co-activator in inositol metabolism in an early yeast study [START_REF] Ebbert | The product of the SNF2/SWI2 paralogue INO80 of Saccharomyces cerevisiae required for efficient expression of various yeast structural genes is part of a high-molecularweight protein complex[END_REF]. Yeast INO80 has now been verified to be the catalytic core of a corresponding huge protein complex, INO80-C, that contains more than 15 subunits and forms an elongated shape [START_REF] Tosi | Structure and Subunit Topology of the INO80 Chromatin Remodeler and Its Nucleosome Complex[END_REF][START_REF] Watanabe | Structural analyses of the chromatin remodelling enzymes INO80-C and SWR-C[END_REF]. INO80-C has also been isolated and analyzed in Drosophila [START_REF] Klymenko | A Polycomb group protein complex with sequence-specific DNA-binding and selective methyl-lysine-binding activities[END_REF] and humans [START_REF] Jin | A mammalian chromatin remodeling complex with similarities to the yeast INO80 complex[END_REF]. Intriguingly, comparison of the complete composition of INO80-C in these organisms has shown that nearly half of its subunits are species specific. Only a few subunits are evolutionarily conserved, which highlights their importance in fundamental INO80-C activities. These conserved subunits include the core remodeler INO80, motor proteins RVB1 and RVB2, actin-related proteins ARP5 and ARP8, and actins [START_REF] Watanabe | The INO80 family of chromatin-remodeling enzymes: regulators of histone variant dynamics[END_REF]. RVB1 and RVB2 belong to the AAA + family of ATPases and regulate the position or modification of nucleosomes [START_REF] Jha | RVB1/RVB2: running rings around molecular biology[END_REF]. ARP proteins possess an actin-fold structure and act as subunits of macromolecular machines that control chromatin remodeling and modification [START_REF] Meagher | Chapter 5. Nuclear actin-related proteins in epigenetic control[END_REF]. Although not identified in Drosophila INO80-C, ARP4, another ARP member, exists in yeast and human INO80-C [START_REF] Watanabe | The INO80 family of chromatin-remodeling enzymes: regulators of histone variant dynamics[END_REF]. Intriguingly, RVB1/RVB2 and ARP4 have also been found to be components -C) in all examined model organisms, which is another Ino80-family complex specialized during evolution for the deposition of histone variant H2A.Z (reviewed in [START_REF] Morrison | Chromatin remodelling beyond transcription: the INO80 and SWR1 complexes[END_REF]. In contrast, the ARP5 subunit is specific to INO80-C in yeast, Drosophila and humans, which points to a tight correlation of their activities. Biochemical analyses have revealed that human ARP5 promotes the binding of the INO80 remodeler to nucleosomes [START_REF] Chen | Multiple modes of regulation of the human Ino80 SNF2 ATPase by subunits of the INO80 chromatin-remodeling complex[END_REF] and couples nucleosome-stimulated INO80 ATPase activity with nucleosome sliding in vitro [START_REF] Willhoft | Synergy and antagonism in regulation of recombinant human INO80 chromatin remodeling complex[END_REF].

Although numerous studies using yeast and human cells have provided insights into the functional relationship of the INO80 remodeler with its distinct subunits, the functional interplay of these proteins during the growth and development of multicellular organisms still remains obscure (reviewed in [START_REF] Gerhold | INO80 and SWR complexes: relating structure to 26 function in chromatin remodeling[END_REF].

T-DNA insertion mutation of plant epigenetic factors can provide an opportunity to unveil their modulatory functions in the context of multicellular organism development. Our previous study as well as the work of other researchers has implicated the Arabidopsis ortholog of INO80 (AtINO80) in somatic homologous recombination and flowering time control [START_REF] Fritsch | The INO80 protein controls homologous recombination in Arabidopsis thaliana[END_REF][START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF].

Although the remodeler AtINO80 has been functionally characterized by the presence of several mutated alleles, little evidence for the existence of a plant INO80-C is currently available. As a result, the participation of presumptive subunits in diverse AtINO80-implicated pathways is still obscure, and their elucidation, especially in the context of plant growth and development, awaits molecular and genetic evidence.

A plant ortholog of ARP5, identified through sequence alignment, has been functionally analyzed in Arabidopsis (AtARP5). AtARP5-defective plants display dwarfed phenotypes with altered organ development and sensitivity to genotoxin [START_REF] Kandasamy | Arabidopsis actin-related protein ARP5 in multicellular development and DNA repair[END_REF]. Even though the growth phenotypes of atino80 and atarp5 single mutants have been independently reported, many fundamental questions still remain unsolved, namely, whether these two proteins physically interact with each other, whether an INO80 complex exists in plants, whether their functions are tightly correlated at the cellular level in the context of plant development, and whether the corresponding growth defects are genetically synergistic or additive. To date, none of these questions have been studied.

Materials and Methods

Plant material and growth conditions

The atino80-5 (SALK_067880), atarp5-2 (GK-386F02) and atarp6-1 (Garlic_599_G03) were derived from the Columbia ecotype. Transgenic plants expressing AtARP6-YFP or EYFP-AtINO80 have been previously reported [START_REF] Qin | ACTIN-RELATED PROTEIN6 Regulates Female Meiosis by Modulating Meiotic Gene Expression in Arabidopsis[END_REF][START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF]. In vitro plant culture was performed on agar-solidified Murashige and Skoog (MS) medium (M0255, Duchefa) supplemented with 0.9% sucrose. Plants were cultivated in the greenhouse at 22°C under 16 h light/8 h dark conditions. For replication stress, seedlings were cultured in the presence of 1 mM hydroxyurea (HU) (H8627, Sigma).

Co-immunoprecipitation (co-IP) assay and mass spectrometry (MS)

The co-IP experiment was performed as previously reported [START_REF] Molitor | Arabidopsis AL PHD-PRC1 Complexes Promote Seed Germination through H3K4me3-to-H3K27me3 Chromatin State Switch in Repression of Seed Developmental Genes[END_REF] by using anti-FLAG antibody (F3165, Sigma) or anti-PRP8 antibody (A303-920A-T, Bethyl). IP proteins and the input fractions were separated on SDS-PAGE and detected by western blotting using anti-FLAG antibody or anti-GFP antibody (A-11122, Invitrogen). For mass spectrometry, the IP proteins were silver stained and cut from gel for further trypsin digestion. The purified peptides were eluted on an analytical capillary column (75 µm×20cm) packed with 2.4-µm spherical C18 reversed-phase material. The eluted peptides were sprayed into a LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) equipped with a nano-ESI ion source.

Database searches were performed on the Mascot server (Matrix Science Ltd., London, UK) against the TAIR database.

Bimolecular fluorescence complementation (BiFC) assay

pXY105 (Yc) and pXY106 (Yn) vectors [START_REF] Sun | The plant-specific transcription factor gene NAC103 is induced by bZIP60 through a new cis-regulatory element to modulate the unfolded protein response in Arabidopsis[END_REF] were used for cloning construction in BiFC assay. Leaves of Nicotiana benthamiana plants were co-infiltrated with Agrobacterium tumefaciens strain GV1301 carrying transgene constructs and/or empty vectors. BiFC fluorescence was observed by using a confocal laser scanning microscope (LSM 710, ZEISS, Germany) at 2-3 days after infiltration.

Microscopy

Seeds were cleared in Hoyer's solution as previously described [START_REF] Schwartz | Disruption of morphogenesis and transformation of the suspensor in abnormal suspensor mutants of Arabidopsis[END_REF]. Rosette leaves were cleared in a solution containing 74% (w/w) chloral hydrate and 7.4% (w/w) glycerol overnight. DIC images of palisade cells was captured by using Nikon ECLIPSE 80i as previously described [START_REF] Zhu | Arabidopsis homologues of the histone chaperone ASF1 are crucial for chromatin replication and cell proliferation in plant development[END_REF]. Images were then analyzed for cell size with Image J (https://imagej.nih.gov/ij/). Root meristem cell number was defined as the number of cortical cells in a file extending the quiescent center (QC) to the first elongated cell as reported in our previous study [START_REF] Ma | Histone chaperones play crucial roles in maintenance of stem cell niche during plant root development[END_REF]. For cortical cell observation, plant roots were immersed in staining solution containing 10 µg/ml propidium iodide (P4170, Sigma) and confocal images were captured using LSM710 microscope. For EdU staining, root tips from plants grown vertically 5 DAG were analyzed as previously described [START_REF] Kotogany | A rapid and robust assay for detection of S-phase cell cycle progression in Plant Cells and tissues by using ethynyl deoxyuridine[END_REF][START_REF] Zhu | Arabidopsis homologues of the histone chaperone ASF1 are crucial for chromatin replication and cell proliferation in plant development[END_REF].

Comet assay

12-day-old plants grown in MS medium supplemented with or without HU were harvested in liquid nitrogen. Comet assay was performed as described in a standard protocol [START_REF] Olive | The comet assay: a method to measure DNA damage in individual cells[END_REF][START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF].

ChIP analysis

ChIP was performed as described in our previous study [START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF]. For each experiment, 1 to 2 g of 12-day-old seedlings was used as one biological replicate, and ChIP analysis was performed in three replicates. The anti-GFP and anti-FLAG antibodies were used at a dilution of 1:100. DNA was analyzed by real-time PCR.

DNase I digestion assay

GST-fusion proteins, histone dimer H2A.Z/H2B and H2A/H2B were expressed and purified as previously reported [START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF][START_REF] Zhu | The Histone Chaperone NRP1 Interacts With WEREWOLF to Activate GLABRA2 in Arabidopsis Root Hair Development[END_REF]. DNase I digestion assay was performed as previously described (Qu et al., 2013)

Results

Protein-protein interaction of AtINO80 with AtARP5

To determine whether AtINO80 interacts with AtARP5, we cloned a genomic fragment containing the full-length AtARP5 gene and introduced a FLAG-encoding DNA segment in front of the start codon, thus enabling the production of an approximately 80-kDa FLAG-AtARP5 fusion protein in planta (Fig. S1). Native promoter-driven expression of FLAG-AtARP5 was able to fully rescue the previously reported phenotype of the atarp5-2 mutant [START_REF] Kandasamy | Arabidopsis actin-related protein ARP5 in multicellular development and DNA repair[END_REF]. We selected stably expressed transgenic plants and crossed them with previously reported EYFP-AtINO80 transgenic plants [START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF] to obtain progeny co-expressing tagged AtINO80 and AtARP5. In a co-IP experiment using these materials, EYFP-AtINO80 was detected in the FLAG-AtARP5 IP fraction (Fig. 1a), indicating their interaction in vivo.

We also performed BiFC experiments. Non-fluorescent N-and C-terminal moieties of YFP fragments (named as Yn and Yc) were fused to full-length AtINO80 and AtARP5 proteins, respectively. Consistent with the co-IP results, only the simultaneous expression of Yn-AtINO80 and Yc-AtARP5 led to YFP fluorescence in the nuclei of tobacco leaf cells, with no YFP signal detected in the negative controls (Fig. S2b, left panel). By using truncated proteins in the same experiment, we demonstrated that the Snf2 domain (AtINO80-∆N; Fig. S2a) is sufficient for the interaction of AtINO80 with AtARP5 (Fig. S2b, right panel).

Next, we conducted a survey of EYFP-AtINO80-and FLAG-AtARP5-associated proteins using immunoprecipitation in combination with mass spectrometry (IP-MS).

According to the IP-MS results, AtINO80 and AtARP5 can efficiently bind to each other. Notably, the list of uncovered AtINO80-associated proteins contained several conserved components of INO80-C in yeast and humans: RVB1, RVB2, ARP5, ARP4 and a series of actin proteins (Table S1). These findings validate the results of our IP-MS experiment, and, at the same time, suggest that these proteins very likely form the conserved part of INO80-C in plants. In addition to these conserved components, more than 200 AtINO80-associated proteins in the IP-MS analysis met our screening criterion (score > 50). Gene Ontology (GO) analysis indicated that these proteins were overrepresented in diverse molecular, cellular and physiological processes (Fig. S3). In contrast, AtARP5 was only associated with 30 proteins; noteworthily, 21 of these proteins were also associated with AtINO80 (Fig. 1b, Table S2). Taken together, our findings indicate that AtINO80 interacts with AtARP5 and can form a larger protein complex in vivo.

AtINO80 and AtARP5 are involved in cell proliferation in leaf development

We next analyzed the genetic interplay of AtINO80 and AtARP5 by crossing the mutant atino80-5 [START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF] with atarp5-2 to obtain the double mutant. As previously described, rosette leaves of both atino80-5 and atarp5-2 mutants were smaller than in the wild type (WT). The retarded growth of the atino80-5 atarp5-2 double mutant relative to the WT resembled that of the atino80-5 mutant and was more severe than that of the atarp5-2 mutant (Fig. 2a).

With respect to the reductions in leaf sizes of the mutants, we looked for defects in cellular proliferation and/or expansion. We quantitatively measured the sizes of palisade cells of first true leaves as previously described [START_REF] Zhu | Arabidopsis homologues of the histone chaperone ASF1 are crucial for chromatin replication and cell proliferation in plant development[END_REF] (Fig. 2b).

Base-10 logarithms of palisade cell surface sizes in all examined leaves followed a nearly normal distribution pattern, but cell size was obviously reduced in all mutants (Fig. 2c). The estimated number of palisade cells per leaf (with the WT set as 100%) were 87%, 64% and 63% lower in the single mutants atarp5-2 and atino80-5 and the double mutant atino80-5 atarp5-2, respectively (Fig. 2d). These physiological data point to the dominant role of AtINO80 in the concerted regulatory functions of the AtINO80-AtARP5 complex in leaf development.

We also examined transcript levels of several key cell-cycle genes. RT-PCR analysis revealed that the transcription of molecular markers in distinct phases of the cell cycle, such as H3.1 (histone), replication-related genes (ORC, CDC6A, CDT1a/b and PCNA1) in the S phase, and the CYCB1;1 gene in the G 2 /M transition, were significantly increased in atino80-5 and atino80-5 atarp5-2 mutants and, to a lesser extent, in atarp5-2 (Fig. 2e). These results suggest that the progression of these phases during the cell cycle was mis-regulated in the mutants. We also measured the primary root elongation of plants grown vertically in MS medium. In parallel with our observations of leaf development, all mutants displayed mild reductions in primary root length (Fig. 3a,b). Arabidopsis roots are composed of meristem, elongation and root-hair zones. The emergence of root hairs in the epidermis marks the onset of cell differentiation and the transition from the elongation zone to the root hair zone [START_REF] Grierson | Root hairs. The Arabidopsis book[END_REF]. Precocious root hair differentiation was observed in all mutants (Fig. 3c), thus indicating that the meristem and elongation zones were collectively restricted. At the same time, we found that root apical meristem (RAM) sizes and meristem cell numbers were significantly reduced in the mutants (Fig. 3c,d), which means that RAM in the mutants could not sustain a sufficient number of meristem cells. We stained the root tips with 5-ethynyl-2′-deoxyuridine (EdU) to visualize cells replicating during the S phase [START_REF] Kotogany | A rapid and robust assay for detection of S-phase cell cycle progression in Plant Cells and tissues by using ethynyl deoxyuridine[END_REF]. More EdU-stained root meristematic cells were observed in roots of the mutants compared with those of the WT (Fig. 3e). Given the reduced meristem cell numbers and increased transcription of S phase-specific genes in the mutants, the increased levels of EdU staining in meristem point to prolongation of the S phase in the impeded cell-cycle progression when AtINO80 and/or AtARP5 are depleted.

AtINO80 and AtARP5 are involved in cell proliferation in primary root

AtINO80-AtARP5 contributes to resistance to DNA damage during replication stress

To provide more insight into the implied function of AtINO80-AtARP5 in S-phase progression, we subjected plants to replication stress. Compared with the WT, all examined mutants exhibited varying degrees of hypersensitivity to hydroxyurea (HU), a DNA replication fork-stalling drug (Fig. 4a). A comparison of weight loss indicated that the hypersensitivity of atino80-5 atarp5-2 to HU was similar to that of atino80-5 and higher than that of atarp5-2 (Fig. 4b).

Defective chromatin replication can lead to instability of replication forks and daughter chromatin [START_REF] Groth | Chromatin challenges during DNA replication and repair[END_REF]. We measured in vivo DNA damage levels using a single-cell gel electrophoresis assay under neutral conditions. In this assay, also known as a comet assay, broken DNA fragments are forced to migrate from agarose-embedded nuclei and thereby form a DNA "comet" tail under electrophoresis [START_REF] Olive | The comet assay: a method to measure DNA damage in individual cells[END_REF]. Remarkably, higher percentages of DNA tails were found under mock conditions in all mutants relative to the WT (Fig. 4c, mock), an observation in agreement with the EdU staining results (Fig. 3e). Replication stress caused by HU treatment led to obviously deteriorated genome stability. Compared with the WT, more DNA fragments were released in plants containing the atino80-5 T-DNA insertion than in the atarp5-2 single mutant, indicative of more DNA fragmentation resulting from potential replication fork collapse (Fig. 4c, HU). We also examined the transcript levels of DNA damage-sensory genes ATM and ATR and DNA-repair genes RAD51 and RAD54. Under both mock and HU treatment conditions, transcript levels of these genes were up-regulated in atino80-5 and atino80-5 atarp5-2 mutants, and, to a lesser extent, in the atarp5-2 mutant when compared to WT (Fig. 4d), which is consistent with the above observations. Collectively, our results indicate that the AtINO80-AtARP5 complex, in which AtINO80 dominates, contributes to chromatin integrity during cell proliferation and prevents genome fragmentation arising from replication stress.

AtARP5 is not involved in AtINO80-mediated flowering control

Our observations of juvenile plants revealed that AtARP5 is in strong functional concert with AtINO80 during vegetative development. In contrast, AtARP5 is clearly not implicated in the AtINO80-mediated vegetative-to-reproductive transition.

Compared with the WT, plants lacking AtARP5 activity exhibited normal flowering times, whereas the atino80-5 atarp5-2 double mutant displayed a late-flowering phenotype reminiscent of atino80-5 (Fig. 5a,b).

AtINO80 binds chromatin regions of the key flowering-repressor gene FLC as well as its paralogs MAF4 and MAF5, thereby down-regulating their transcript levels [START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF]. RT-PCR analysis of 12-day-old seedlings revealed that FLC/MAF4/MAF5, concomitant with flowering times, were up-regulated in the atino80-5 atarp5-2 double mutant at levels similar to atino80-5 but were not significantly affected in atarp5-2 (Fig. 5c).

We next performed a ChIP analysis to compare the detailed chromatin distribution of these two proteins along the FLC gene. On the basis of our previous study (Zhang showed a low affinity to most examined FLC chromatin regions relative to the reference gene ACT2, and no peaks were observed (Fig. 5d). A similar discrepancy in EYFP-AtINO80 and FLAG-AtARP5 distributions was found in consecutive chromatin regions of MAF4 and MAF5 (Fig. S4).

To obtain more evidence, we selected additional genes exhibiting transcript level mis-regulation in atino80-5 (fold-change > 2). These genes, which included DDF1 (DWARF AND DELAYED FLOWERING 1), EDA29 (EMBRYO SAC DEVELOPMENT ARREST 29), LEA (LATE EMBRYOGENESIS ABUNDANT PROTEINS RELATED), AGL24 (AGAMOUS-LIKE 24) and HAI1 (HIGHLY ABA-INDUCED PP2C GENE 1), have roles related to various plant-specific development stages. Except for AGL24, whose transcription in atarp5-2 remained at WT levels, transcriptional changes of these examined genes were consistent between atino80-5 and atarp5-2 (Fig. S5a). In contrast, the distributions of AtINO80 and AtARP5 in 5′-and middle regions of the AGL24 gene were significantly different. A similar discrepancy was also found in individual examined regions of DDF1 and EDA29 (Fig. S5b). These findings support a model in which AtINO80-AtARP5 co-enrichment is not obligatory in all regions of AtINO80 target genes.

Local enrichment of AtINO80 within FLC/MAF4/MAF5 is affected by AtARP6 activity

AtINO80 can interact with histone variant H2A.Z and moderately enhance H2A.Z peaks within FLC/MAF4/MAF5 chromatin regions [START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF]. AtARP6, the important subunit of Arabidopsis SWR1-C, is responsible for replacing the canonical H2A with the variant H2A.Z in nucleosomes, with levels of chromatic H2A.Z accordingly found to be drastically reduced in the AtARP6-deficient mutant atarp6-1 [START_REF] Deal | Repression of flowering in Arabidopsis requires activation of FLOWERING LOCUS C expression by the histone variant H2A[END_REF][START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF]. We thus wondered whether AtARP6 has an effect on local AtINO80 accumulation. Transgenic plants harboring EYFP-AtINO80 were introgressed into an atarp6-1 background. Intriguingly, the IP efficiency of EYFP-AtINO80 in consecutive FLC chromatin regions 8-12, but not in other regions or the reference gene ACT2, was significantly enhanced in atarp6-1 compared with the AtARP6 +/+ background (Fig. 5d). Similar accumulation patterns were also observed in the chromatin regions of MAF4 and MAF5 (Fig. S4) as well as EDA29 (Fig. S5b). To test the possibility that AtARP6 directly alters AtINO80 accumulation through protein-protein interaction, we included native promoter-driven AtARP6-YFP [START_REF] Qin | ACTIN-RELATED PROTEIN6 Regulates Female Meiosis by Modulating Meiotic Gene Expression in Arabidopsis[END_REF] in the same ChIP experiments. AtARP6-YFP displayed chromatin enrichment peaks near both ends of FLC/MAF4/MAF5 (Fig. 5d and S4), which are spatially separate from those of AtINO80. In addition, we detected no AtINO80-AtARP6 interaction in a pull-down assay (Fig. S7b) nor by IP-MS. Our findings imply that AtARP6 activity (histone turnover), rather than the AtARP6 protein itself, may have an unexpected relationship with the local chromatin recruitment of AtINO80.

H2A.Z/H2B can significantly inhibit the DNA-binding capability of AtINO80-N

The N-terminal moiety of the AtINO80 protein contains a conserved DBINO domain in its primary sequence. In humans, this domain is thought to bind DNA [START_REF] Bakshi | Characterization of a human SWI2/SNF2 like protein hINO80: demonstration of catalytic and DNA binding activity[END_REF]. We therefore expressed and purified GST-fused AtINO80-N, a truncated protein that starts from the N-terminus and ends before the intact ATPase domain. AtINO80-N contains both a DBINO domain and several ΦxxΦΦ motifs that can preferentially bind histone variant dimers H2A.Z/H2B [START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF] (Fig. S6a,c).

We performed a DNase I digestion assay as previously described (Qu et al., 2013) using full-length FLC genomic DNA as an input (DNA #1, set as 100%) (Fig. 6a).

DNase I was able to completely digest the naked DNA template (DNA #2), with no remaining DNA detected in a subsequent quantitative PCR (Fig. 6b). The addition of AtINO80-N was able to partially protect the naked DNA from DNase I digestion (DNA #3), thus indicating that AtINO80 through its N-terminal moiety can weakly bind and thus shield DNA. In this experiment, AtINO80-N exhibited scarcely any sequence preference in terms of local DNA shielding (Fig. 6b). Similar results were also obtained using full-length MAF4 and MAF5 as DNA templates. Next, we purified H2A.Z/H2B and H2A/H2B dimers as reported in our previous study [START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF]. We premixed these dimers with AtINO80-N and repeated the DNase I digestion experiment (DNA #4). Intriguingly, the addition of H2A.Z/H2B significantly weakened the ability of AtINO80-N to bind DNA in vitro, and the inhibition of DNA binding was dramatically increased when the molar ratio of H2A.Z/H2B to AtINO80-N was incremented. In comparison, H2A/H2B with an even high molar ratio to AtINO80-N than that of H2A.Z/H2B had a much weaker inhibitory effect on the DNA-binding ability of AtINO80-N (Fig. 6c). These in vitro findings that the DNA-binding capability of AtINO80-N is impaired when bound to a preferential histone substrate may help to explain the significantly decreased local AtINO80 chromatin enrichment of FLC observed in the presence of AtARP6 activity, which replaces H2A with H2A.Z.

AtINO80 interacts with the core spliceosome component PRP8 and sustains the proper polyadenylation of COOLAIR at the proximal site

As the AtINO80 DBINO domain showed little sequence specificity in vitro, the recruitment of AtINO80 to FLC is likely mediated via other pathway(s). Taking the IP-MS results into consideration, we thus investigated the relevance of AtINO80-associated proteins to FLC transcriptional regulation. In fact, the protein in the IP-MS list with the second highest protein score (1,488) was PRP8 (PRE-MRNA PROCESSING 8), a core spliceosome component that is critical to FLC antisense RNA COOLAIR processing [START_REF] Marquardt | Functional consequences of splicing of the antisense transcript COOLAIR on FLC transcription[END_REF].

Using an antibody specific to endogenous PRP8, we verified the interaction of this protein with EYFP-AtINO80 through a co-IP experiment (Fig. S7a). PRP8 is a huge protein (MW > 270 kDa) and contains an N-terminal PROCN domain, a middle RNA-recognizing RRM domain, a C-terminal RNase H domain and a MOV34 domain (Fig. S6b). Marquardt et al. have reported that prp8-6, a weak mutant allele with a point mutation in its RNase H domain, exhibits reduced usage of the proximal site (PAP) of the COOLAIR poly(A) and an increased usage of the distal site (PAD).

Intriguingly, our RT-PCR analysis revealed that COOLAIR PAP usage was also reduced in an atino80-5 background, while transcript levels of most major RNA processing factors remained relatively normal (Fig. S8). We thus selected for expression the GST-fusion proteins of PROCN (GST-PRP8-PROCN) and the combination of RNase H and MOV34 domains (named as GST-PRP8-R/M) (Fig. S6b,c). In the pull-down assay, the positive control GST-AtARP5, but not GST-ARP6, was able to bind EYFP-AtINO80. Intriguingly, GST-PRP8-R/M, but not GST-PRP8-PROCN, was also able to interact with EYFP-AtINO80, thereby providing more evidence for the protein-protein interaction of PRP8 with AtINO80 (Fig. S7b). Because the reduced use of the PAP in antisense COOLAIR disrupted transcriptional repression of sense FLC gene [START_REF] Liu | Targeted 3' processing of antisense transcripts triggers Arabidopsis FLC chromatin silencing[END_REF], our current findings are consistent with the FLC de-repression in atino80-5 reported in our previous study [START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF]. These results also provide further insights into the molecular mechanism of AtINO80 in local transcriptional regulation.

The synergistic function of AtINO80 and AtARP6 in plant growth and development

Next, we crossed atino80-5 with atarp6-1 to determine their genetic relationship.

Compared with individual atino80-5 and atarp6-1 plants, the double mutant identified in the F 2 segregating population displayed an obviously weak phenotype, i.e., greatly dwarfed plants. Under our growth conditions, most identified atino80-5 atarp6-1 double mutants generated only one or two inflorescences over their lifespans.

Although these inflorescences produced flowers, most of the latter (above 80%, n > 25) did not develop into siliques (Fig. 7a). Any generated seeds were carefully collected for subsequent germination. The germination rate of both atino80-5 and atarp6-1 seeds was nearly 100%, similar to that of the WT. In contrast, fewer than 15% of collected double mutant seeds in the F 3 generation successfully germinated under our growth conditions. Nevertheless, these surviving seedlings displayed dramatically retarded growth compared with individual single mutants or the WT (Fig. 7b). The consequent dramatic reduction in plants and seeds in successive generations hindered our ability to obtain a sufficient pool of genetic materials for downstream molecular and functional analyses.

When we dissected aborted seeds of the atino80-5 atarp6-1 double mutant (n > 100), we found that the embedded embryos were arrested in various stages.

Approximately 4% of arrested embryos were at the early heart stage, with approximately 26% stuck at the torpedo stage and 42% remaining at the phase of cotyledon embryo formation. Only 28% of embryos appeared in a nearly mature form, but these seeds still failed to germinate (Fig. 7c). The severely curtailed phenotype and poor fertility of the atino80-5 atarp6-1 double mutant clearly demonstrates that AtINO80 and AtARP6 act in synergy (beyond their regulation of flowering-repressor genes and flowering time) to exert crucial roles in embryogenesis and post-embryonic development of both vegetative and floral organs. The functional cooperation of these two genes is thus required for diverse stages of plant growth and development.

Coordination of AtARP5 and AtARP6 in chromatin stability

Unlike atino80-5 atarp6-1, the viability and fertility of the atarp5-2 atarp6-1 double mutant was comparable to that of the individual single mutants and the WT.

The apparent growth phenotype of atarp5-2 atarp6-1 at 12 DAG was a slight reduction in leaf size and upward-curled leaves (Fig. 8a). Indicative of their synergistic role in leaf morphology, the severity of such defects was increasingly obvious in older plants growing in soil at 25 DAG (Fig. 8b). In comparison, the simultaneous loss of function in AtARP5 had no significant effect on the previously reported early-flowering phenotype of atarp6-1 (Fig. 8c) [START_REF] Deal | Repression of flowering in Arabidopsis requires activation of FLOWERING LOCUS C expression by the histone variant H2A[END_REF], an observation in line with the dispensable role of AtARP5 in AtINO80-mediated flowering regulation. The great contrast in phenotypes between atino80-5 atarp6-1 and atarp5-2 atarp6-1 reflects the distinct significance and roles of AtINO80 and AtARP5 in SWR1-C-associated key developmental transitions and plant growth stages and provides more evidence of the dominant role of AtINO80 as a remodeler in the protein complex.

We also wondered whether chromatin instability in the absence of the AtINO80-AtARP5 complex (Fig. 4c) would be further aggravated by the simultaneous depletion of AtARP6. Because plant materials of atino80-5 atarp6-1 were limited, we performed a comet assay using the atarp5-2 atarp6-1 double mutant.

Under normal growth conditions, a higher percentage of DNA in tails was found in atarp5-2 atarp6-1 than in individual single mutants (Fig. 8d). To unravel the molecular response to the absence of AtARP5 and AtARP6 activities, we examined transcript levels of DNA damage-sensory genes ATM/ATR/PARP2 and repair genes [START_REF] Han | Roles and activities of chromatin remodeling ATPases in plants[END_REF]. In this study, we have proved that the remodeler AtINO80 can interact with AtARP5 in vivo. AtINO80-associated proteins uncovered in addition to AtARP5 by our IP-MS analysis included RVB1, RVB2, ARP4 and actin proteins, which collectively constitute a putative minimal core complex of INO80-C in plants similar to its yeast and human counterparts [START_REF] Willhoft | Synergy and antagonism in regulation of recombinant human INO80 chromatin remodeling complex[END_REF]. Moreover, most AtARP5-associated proteins overlapped with those of AtINO80, indicative of a larger plant protein complex. Taken together, our study findings have established a molecular basis for the functional characterization of the coordination of AtINO80 with its subunits.

Our previous research on a single mutant of AtINO80 focused on the important role of that protein in somatic homologous recombination and flowering timing control [START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF]. In the present study, we investigated the concerted roles of AtINO80 with its subunit AtARP5 in cell proliferation during plant leaf and root development. Detailed analyses of root apical meristem size and meristem cell number clearly demonstrated that sufficient numbers of meristem cells could not be sustained in the meristem zone of the mutants. At the same time, EdU staining revealed that more meristematic cells were arrested during the DNA synthesis phase in the absence of AtINO80-AtARP5, especially under normal growth conditions. This result suggests that the defects of AtINO80-C-deficient cells are more likely associated with traversal of the S phase rather than entry into it. Exogenous replication stress can intensify the collapse of an ongoing replication fork [START_REF] Groth | Chromatin challenges during DNA replication and repair[END_REF], and more DNA fragmentation was detected in young seedlings depleted of the protein complex. The results of our molecular, cellular and genetic analyses on atino80/atarp5 mutants point to the important roles of AtINO80-AtARP5 in ensuring 2008;[START_REF] Shimada | Ino80 chromatin remodeling complex promotes recovery of stalled replication forks[END_REF][START_REF] Vincent | ATP-dependent chromatin remodeling shapes the DNA replication landscape[END_REF][START_REF] Falbo | Involvement of a chromatin remodeling complex in damage tolerance during DNA replication[END_REF]. However, replication origins of large plant genomes have many features that are distinct from those in the small yeast genome, such as no DNA consensus sequence at origin sites and a strong relationship between the chromatin landscape and origin specification and genome replication (reviewed in [START_REF] Sequeira-Mendes | Links between genome replication and chromatin landscapes[END_REF]. In the context of multicellular organism development, the activities of plant INO80 in the cell cycle thus seemingly cannot be explained by simply invoking the above-mentioned mechanism in yeast. In addition, yeast INO80 has been reported to be dispensable for replication fork stability after treatment with HU [START_REF] Falbo | Involvement of a chromatin remodeling complex in damage tolerance during DNA replication[END_REF]. This finding is inconsistent with our observation of increased numbers of DNA fragments in the comet assay, thus implying a distinct role for AtINO80 (and its complex) in maintaining stalled replication forks.

In human cells, BRCA1-associated protein-1, a de-ubiquitinating enzyme, has been recently reported to stabilize INO80 through de-ubiquitination and to recruit this remodeler to replication forks based on interaction with ubiquitinated H2A [START_REF] Lee | Stabilization and targeting of INO80 to replication forks by BAP1 during normal DNA synthesis[END_REF]. In our study, however, we found no Arabidopsis ortholog of BAP1 or any de-ubiquitinating enzymes identified as AtINO80-associated proteins in the IP-MS analysis. Our study findings raise the possibility that AtINO80 may participate in chromatin duplication and cellular proliferation in a plant-specific pathway. Unlike growth in animals, post-embryonic plant growth must be flexible to allow development that can adapt to changing environmental stimuli. Future analysis of AtINO80-associated proteins other than AtARP5 and their functional coordination should provide novel insights into the molecular mechanism underlying the participation of chromatin-remodeling factors in plant growth.

Role of AtARP5 as a subunit of AtINO80

A fixed composition can generally be taken for granted when considering chromatin-remodeling complexes, as such an arrangement promotes steady and continuous activity. Nevertheless, the presence of AtARP5 as a subunit of AtINO80 is apparently not obligatory during some key developmental stages. In particular, our ChIP analysis did not uncover similar patterns of AtARP5 and AtINO80 distribution in chromatin regions of the flowering-repressor genes FLC/MAF4/MAF5, an observation consistent with the dispensable role of AtARP5 in AtINO80-mediated transcriptional regulation and corresponding flowering control. We thus propose that AtINO80 may exert its physiological function with its subunits in a flexible manner instead of continually in the form of a holo-complex.

Even in yeast, a unicellular organism, the contribution of ARP5 to INO80-C seems to be fluid, with association or dissociation as well as consequent modulation of chromatin-remodeling function found to be dynamic. Sucrose gradient separation of yeast extracts has revealed a separate sub-complex containing a much higher quantity of ARP5 relative to INO80-C [START_REF] Yao | The INO80 Complex Requires the Arp5-Ies6 Subcomplex for Chromatin Remodeling and Metabolic Regulation[END_REF]. This result suggests that a substantial pool of free ARP5 exists in vivo that can act, if needed, as a separable module for the yeast remodeler INO80. Moreover, only a slight difference in chromatin distribution has been found between yeast INO80 and ARP5, but, intriguingly, this differential intensity in chromatin occupancy has functional consequences [START_REF] Yen | Genome-wide Nucleosome Specificity and Directionality of Chromatin Remodelers[END_REF].

Besides the different functions of AtARP5 and AtINO80 in flowering time control and local transcriptional regulation, we also noticed that a small fraction (9/30) of AtARP5-associated proteins do not overlap with those of AtINO80, implying that AtARP5 may possess uncharacterized AtINO80-independent activities. We cannot yet exclude the possibility, however, that the atarp5 single mutant will display atino80-irrelevant phenotypes under certain growth conditions or stresses in future analyses.

Interplay between SWR1-C and INO80-C

AtINO80 preferentially interacts with histone variant H2A.Z [START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF], and H2A.Z/H2B addition can efficiently inhibit the DNA-binding capability of AtINO80-N in vitro. Moreover, the local chromatin recruitment of AtINO80 in several of its examined target genes was significantly enhanced in the absence of AtARP6, (unique to SWR1-C) in IP-MS and pull-down assays. In addition, the chromatin distribution of AtARP6 was distinct from that of AtINO80/AtARP5 in examined chromatin regions, which suggests that any spatial interference between these two remodeling complexes would have a minimal effect on local transcriptional regulation.

We propose that dynamic H2A.Z more likely functions as a molecular bridge to connect their activities. In fact, evidence from yeast also linked INO80 activities to pre-existing histone variants or modifications. Yeast INO80 can mobilize H2A.Z-containing nucleosomes more efficiently than those containing canonical H2A [START_REF] Brahma | INO80 exchanges H2A.Z for H2A by translocating on DNA proximal to histone dimers[END_REF]. INO80 can also be recruited to induce DSB sites through specific interaction with γ-H2A.X, a phosphorylated derivative adjacent to the damaged sites [START_REF] Morrison | INO80 and gamma-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair[END_REF]. Collectively, these findings support the notion that specific chromatin feature(s) may guide INO80 for its proper anchorage or dissociation as well as its subsequent output of remodeling activities.

INO80-C in yeast and humans has a wide range of activities beyond H2A.Z turnover, such as nucleosome eviction and nucleosome spacing establishment (reviewed in [START_REF] Gerhold | INO80 and SWR complexes: relating structure to 26 function in chromatin remodeling[END_REF]. Consequently, the molecular mechanism underlying the activity of INO80 in local transcriptional regulation in plants is not likely associated exclusively with the H2A.Z variant. Consistent with this idea, we found that the chromatin distribution of AtINO80 in many target genes (DDF1/LEA/AGL24/HAI1) was not affected by the absence of AtARP6. In this study, we also observed protein-protein interaction between AtINO80 and COOLAIR-targeting PRP8; however, a null mutation in PRP8 leads to embryonic lethality [START_REF] Schwartz | Disruption of morphogenesis and transformation of the suspensor in abnormal suspensor mutants of Arabidopsis[END_REF]. Whether AtINO80 recruitment and subsequent activities on its target genes is relevant to PRP8 or other sequence-specific associated proteins is still largely unknown. Multiple factors or pathways, rather than a single one, may act together to determine the chromatin recruitment of AtINO80 to its diverse target genes. To resolve this question, further systematic investigation and more evidence is needed. , 2015). In plants, AtINO80 and core components of AtSWR1-C have in fact been independently proven to play important roles in DNA repair, especially in the repair of DSBs in somatic or reproductive cell lineages [START_REF] Rosa | The Arabidopsis SWR1 Chromatin-Remodeling Complex Is Important for DNA Repair, Somatic Recombination, and Meiosis[END_REF][START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF]. In this study, our molecular and genetic analyses based on atarp5-2 atarp6-1 and partially on atino80-5 atarp6-1 supported the conserved synergy of these two remodeling complexes in chromatin stability during plant growth.

In contrast to the crucial role of INO80 in Drosophila and mouse embryonic development [START_REF] Bhatia | Chromatin remodeling protein INO80 has a role in regulation of homeotic gene expression in Drosophila[END_REF][START_REF] Wang | INO80 Facilitates Pluripotency Gene Activation in Embryonic Stem Cell Self-Renewal, Reprogramming, and Blastocyst Development[END_REF][START_REF] Qiu | Ino80 is essential for proximal-distal axis asymmetry in part by regulating Bmp4 expression[END_REF], AtINO80 is relatively unimportant at this stage [START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF]. This large discrepancy in the importance of INO80 between Arabidopsis and animals raises the hypothesis that the function of AtINO80 may be redundant with that of other factors during very early development in plants. In this study, in the atarp6-1 background, the defective embryogenesis observed in the atino80-5 mutant was far more severe compared with atarp5-2. This disparity cannot be simply attributed to their activities in the presence of AtARP6, with only mild differences observed in cellular proliferation and maintenance of genome integrity. Our genetic analysis of atino80-5/atarp6-1 has revealed the complexity of functional coordination between the AtINO80 remodeler (d) Estimated palisade cell number per leaf according to previous study [START_REF] Zhu | Arabidopsis homologues of the histone chaperone ASF1 are crucial for chromatin replication and cell proliferation in plant development[END_REF]. The ratios of mean values in mutants to that of WT are shown.

  report the interaction of AtINO80 and AtARP5 and their associated proteins. AtARP5 acts as an accessory to AtINO80 in cellular proliferation and plant response to replication stress, but does not participate in the flowering transition or AtINO80-mediated transcriptional modulation of key flowering-repressor genes FLC/MAF4/MAF5. We unexpectedly found that the recruitment of AtINO80 to FLC/MAF4/MAF5 is affected by AtARP6, the specific and important component of SWR1-C. Genetic crosses between mutants of atino80/atarp6 and atarp5/atarp6 revealed the synergistic and crucial roles of these two chromatin-remodeling complexes in diverse stages of plant growth and in genome stability maintenance.

  ., 2015), we redesigned primer pairs (n = 18), one or two nucleosomes at a time, to allow coverage of the entire gene region and terminator of FLC (approximately 7.3 kb) (Fig.5d). Our results confirmed the chromatin recruitment of EYFP-AtINO80 in FLC observed in the previous study, which slightly peaked near the first-intron/second-exon boundary. In comparison, co-expressed FLAG-AtARP5

  . All of these tested genes were synergistically up-regulated in the atarp5-2 atarp6-1 double mutant (Fig.8e). Taken together, these results suggest that AtARP5 and AtARP6 act in concert to maintain chromatin integrity during plant growth.Plants require precise control of dynamic chromatin structure at diverse growth stages and transitions. Analysis of the functional coordination of chromatin-remodeling factors in protein complexes and identification of their associated subunits is very important for understanding the molecular mechanisms controlling their action during plant growth and development (reviewed in

  progression and maintaining chromatin integrity under replication stress.Strong evidence has been uncovered for the S phase-specific association of INO80 with origins of replication in the yeast genome (Papamichos-Chronakis &Peterson,

  which encodes the specific component of Arabidopsis SWR1-C mediating H2A.Z turnover. Although several AtINO80-associated proteins detected in this study have also been included in the inferred plant SWR1-C (reviewed in March-Diaz & Reyes, we did not detect any interaction between AtINO80/AtARP5 and AtARP6

  Studies in yeast and human cells have provided abundant evidence that INO80-C and SWR1-C synergistically function as genomic guardians to maintain genomic integrity in response to DNA damage (reviewed in[START_REF] Morrison | Chromatin remodelling beyond transcription: the INO80 and SWR1 complexes[END_REF] Gerhold

  and AtARP6 (unique to SWR1-C), and highlighted their synergistic roles in key fundamental biological processes. More challenging analyses are required to unravel the complicated mechanisms responsible for the observed embryogenetic defects, which should shed light on the critical process of chromatin modulation during very early plant developmental stages, an important topic in basic plant science and crop research.

  Figure legends

Fig. 2

 2 Fig. 2 Double mutant analysis of atino80-5/atarp5-2 in leaf development.(a) Wild-type (WT), atino80-5, atarp5-2 and the double mutant atino80-5 atarp5-2 plants grown on soil at 25 DAG (days after germination). Bar = 10 mm. (b) Representative DIC (Differential Interference Contrast) images of palisade cells. Bar = 100 µm.

  Comparison of the cell size of palisade cells (log µm 2 ). Each distribution curve was derived from a total of over 300 palisade cells from 15 first true leaves of plants in(a).

  (e) Quantitative RT-PCR analysis of cell cycle and DNA replication-related genes. The analyses were performed using 12-day-old seedlings in three independent experiments. ACT2 was used as reference gene. Mean values of the relative gene expression levels in the mutants compared to WT (set as 1.0) are shown together with error bars indicating standard deviations. Asterisks indicate statistically significant difference (P < 0.05) in mutants when compared with WT.

Fig. 3

 3 Fig. 3 Double mutant analysis of atino80-5/atarp5-2 in root development.

  (a) WT, atino80-5, atarp5-2 and the double mutant atino80-5 atarp5-2 plants grown vertically on MS medium at 12 DAG. Bar = 10 mm. (b) Comparison of root length. The mean value from 20 plants is shown with error deviations. Asterisks indicate statistically significant difference (P < 0.05) in mutants when compared with WT. (c) The precocity of root hair differentiation occurred in all the mutants. DIC images were taken on roots at 5 DAG. Yellow triangles indicate the emergence of root hair in epidermis. Red lines mark the root meristem zones. Bar = 20 µm. (d) Comparison of root meristem cell number at 5 DAG. Root meristem cell number was counted as previously reported (Ma et al., 2018). The mean value from 15 plants is shown with error bars representing standard deviations. Asterisks indicate statistically significant difference (P < 0.05) in mutants when compared with WT. (e) Comparison of EdU-staining on root tips at 5 DAG. Bar = 1 mm.

Fig. 4

 4 Fig. 4 AtINO80-AtARP5 contributes to DNA damage resistance to replication stress.(a) Images of 12-day-old plants grown on MS medium (Mock, upper panel) or medium containing 1mM HU (HU treatment, lower panel). Bar=10 mm. (b) Net weight quantification of 10 seedlings grown in (a). Mean values are shown with error bars from ten biological replicate measurements. Asterisks indicate

Fig. 5

 5 Fig. 5 AtARP5 is not involved in AtINO80-mediated flowering control. (a) Photos showing WT, atino80-5, atarp5-2 and the double mutant atino80-5 atarp5-2 plants grown in soil at 40 DAG. (b) Days to bolting (upper) and rosette leaf numbers (lower) (n > 30 each). Bars represent the standard deviations. Asterisk indicates statistically significant difference (P < 0.05). (c) Relative expression level of flowering-related genes in 12-day-old seedlings. ACT2 was used as reference gene. Mean values are shown with error bars from three independent experiments. Asterisk indicates statistically significant difference (P <

Fig. 6

 6 Fig. 6 H2A.Z/H2B can greatly inhibit the DNA-binding activity of AtINO80-N in vitro. (a) Schematic representation of DNase I digestion experiment (DNA #1-#4). (b) The percentages of DNA remaining were revealed by the subsequent quantitative PCR. Data from DNA #1 (no DNase I digestion, black columns) was set as 100%. Mean values are shown together with error bars indicating standard deviations. Note that DNA#2 (white columns) cannot be shown since DNA was fully digested by DNase I. (c) The percentages of DNA remaining (in DNA #4) after DNase I digestion in

  region 10 of FLC (since AtINO80-N has little sequence preference in the whole examined regions). Histone dimer H2A.Z/H2B (or H2A/H2B) was premixed with GST-AtINO80-N in the indicated molar ratio and the other procedures were the same as DNA #3. Mean values are shown with error bars indicating standard deviations.

Fig. 7

 7 Fig. 7 Genetic interplay between AtINO80 and AtARP6.

  (a) Comparison of inflorescence of WT and each mutant at F 2 segregation. Bar=10 mm. (b) Comparison of 12-day-old seedling of WT and each mutant at F 3 generation. Note that only germinated atino80-5 atarp6-1 double mutant were shown. Bar=10 mm. (c) Embryogenesis was arrested at distinct stages in atino80-5 atarp6-1. Percentage of arrested embryos in each stage is shown (n>100). Bar=100 µm.

Fig. 8

 8 Fig. 8 Functional synergy of AtARP5 and AtARP6 in genome stability. (a) Image of 12-day-old plants grown on MS medium. Bar=10 mm. (b) Comparison of first true leaf from 25-day-old plants. Bar=10 mm. (c) Comparison of flowering time and rosette leaf number. (d) Nuclear DNA lesion levels revealed in comet assay. The mean value of more than 200 nuclei was shown together with error bar. The comparison of the percentage of DNA in tail between the WT and mutant plants grown on the normal medium was shown. Asterisks indicate statistically significant difference (P < 0.05) of mutants in comparison with WT.(e) Relative expression levels of DNA damage sensory and repair genes in WT (set as 1) and mutant plants. ACT2 was used as reference gene. The analyses were performed

Manuscript submitted to New Phytologist for review

Acknowledgements

We thank Professor Yuan Qin for the seeds of transgenic plant AtARP6-YFP. This research work was conducted within the context of the International Associated Laboratory Plant Epigenome Research, LIA PER. It was financially supported by the National Natural Science Foundation of China (Grants NSFC 31671341).

Table S1 The conserved components of INO80-C found in AtINO80-associated proteins.

Table S2

The list of 21 overlapped AtINO80-and AtARP5-associated proteins (score>50).