Chao Li

Yuhao Liu

Wen-Hui Shen

Yu Yu

Aiwu Dong
email: aiwudong@fudan.edu.cn

Chromatin-remodeling factor OsINO80 is involved in regulation of gibberellin biosynthesis and is crucial for rice plant growth and development

Keywords: rice; gibberellin, chromatin-remodeling factor, transcriptional regulation

The phytohormone gibberellin (GA) plays essential roles in plant growth and development.

Here, we report that OsINO80, a conserved ATP-dependent chromatin-remodeling factor in rice (Oryza sativa), functions in diverse biological processes and GA biosynthesis. OsINO80knockdown mutants, derived from either T-DNA insertion or RNA interference, display typical GA-deficient phenotypes, including dwarfism, reduced cell length, late flowering, retarded seed germination and impaired reproductive development. Consistently, transcriptome analyses reveal that OsINO80 knockdown results in down-regulation by more than two-fold of over 1000 genes, including the GA biosynthesis genes CPS1 and GA3ox2, and the dwarf phenotype of OsINO80-knockdown mutants can be rescued by the application of exogenous GA3. Chromatin immunoprecipitation (ChIP) experiments show that OsINO80 directly binds to the chromatin of CPS1 and GA3ox2 loci. Biochemical assays prove that OsINO80 specially interacts with histone variant H2A.Z and the H2A.Z enrichments at CPS1 and GA3ox2 is decreased in OsINO80-knockdown mutants. Thus, our study identified a new rice chromatin-remodeling factor, OsINO80, and demonstrated that OsINO80 is involved in regulation of the GA biosynthesis pathway and plays critical functions at many aspects of rice plant growth and development.

INTRODUCTION

Gibberellins (GAs) are important endogenous phytohormones and modulate diverse plant developmental processes, including seed germination, leaf and stem elongation, flowering transition, and reproductive development [START_REF] Hedden | A Century of Gibberellin Research[END_REF]. By searching for GAdeficient mutants, the key components in the GA pathway, including seven types of enzymes responsible for GA biosynthesis and metabolism, have been identified from both Arabidopsis thaliana and rice. The enzymes comprise ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA 20-oxidase (GA20ox), GA 3-oxidase (GA3ox), and GA 2-oxidase (GA2ox) [START_REF] Sakamoto | An Overview of Gibberellin Metabolism Enzyme Genes and Their Related Mutants in Rice[END_REF][START_REF] Sun | Gibberellin Metabolism, Perception and Signaling Pathways in Arabidopsis[END_REF]).

In addition, knowledge has also been gained on some important regulators mediating GA signal transduction. It is known that GIBBERELLIN INSENSITIVE DWARF1 (GID1) proteins act as GA receptors, while Arabidopsis DELLA and rice SLENDER RICE1 (SLR1) are negative regulators of GA signaling [START_REF] Ueguchi-Tanaka | Gibberellin Receptor and Its Role in Gibberellin Signaling in Plants[END_REF]. Dwarfism is the typical phenotype of mutants defective in GA synthesis or signaling. In addition, GA-deficient plants often exhibit dark-green leaves, retarded growth, late flowering, and sterility [START_REF] Sakamoto | An Overview of Gibberellin Metabolism Enzyme Genes and Their Related Mutants in Rice[END_REF][START_REF] Sun | Gibberellin Metabolism, Perception and Signaling Pathways in Arabidopsis[END_REF].

GA biosynthesis and signaling pathways allow plants rapidly responding to environmental changes, such as light, temperature, biotic and abiotic stresses [START_REF] Yamaguchi | Gibberellin Metabolism and its Regulation[END_REF]). Thus, GArelated genes tend to be precisely regulated during plant growth and development [START_REF] Yamaguchi | Gibberellin Metabolism and its Regulation[END_REF]. In recent years, several studies in Arabidopsis point to a link between the GA pathway and the chromatin-remodeling regulatory mechanism. There are four types of chromatinremodeling factors in eukaryotes, the Swi/Snf, Iswi, Chd, and Ino80 families [START_REF] Clapier | The biology of chromatin remodeling complexes[END_REF]. BRAHMA (BRM), the ATPase subunit of Swi/Snf-type chromatin-remodeling complexes, is a positive regulator of GA-mediated responses in Arabidopsis [START_REF] Archacki | BRAHMA ATPase of the SWI/SNF Chromatin Remodeling Complex Acts as a Positive Regulator of Gibberellin-Mediated Responses in Arabidopsis[END_REF]). SWI3C, a core component of the Arabidopsis Swi/Snf-type chromatin-remodeling complexes, physically interacts with DELLA proteins, and this interaction is necessary for proper GA biosynthesis and signaling [START_REF] Sarnowska | DELLA-Interacting SWI3C Core Subunit of Switch/Sucrose Nonfermenting Chromatin Remodeling Complex Modulates Gibberellin Responses and Hormonal Cross Talk in Arabidopsis[END_REF]. The Chd-type chromatinremodeling factor PICKLE (PKL) also interacts with DELLA proteins and functions in Arabidopsis GA-dependent responses [START_REF] Henderson | PICKLE Acts throughout the Plant to Repress Expression of Embryonic Traits and May Play a Role in Gibberellin-Dependent Responses[END_REF][START_REF] Zhang | The Chromatin-Remodeling Factor PICKLE Integrates Brassinosteroid and Gibberellin Signaling during Skotomorphogenic Growth in Arabidopsis[END_REF]. The Iswi-type CHROMATIN REMODELING 11 (CHR11) and CHR17 have been shown to be involved in Arabidopsis flowering transition, reproductive development, and genome-wide nucleosome distribution [START_REF] Huanca-Mamani | CHR11, a chromatin-remodeling factor essential for nuclear proliferation during female gametogenesis in Arabidopsis thaliana[END_REF][START_REF] Li | Imitation Switch chromatin remodeling factors and their interacting RINGLET proteins act together in controlling the plant vegetative phase in Arabidopsis[END_REF][START_REF] Li | ISWI proteins participate in the genome-wide nucleosome distribution in Arabidopsis[END_REF], and the Ino80-family members PHOTOPERIOD-INDEPENDENT EARLY-FLOWERING 1 (PIE1) and AtINO80 have been reported to play crucial roles in Arabidopsis genome stability and plant development [START_REF] Noh | PIE1, an ISWI family gene, is required for FLC activation and floral repression in Arabidopsis[END_REF][START_REF] Fritsch | The INO80 protein controls homologous recombination in Arabidopsis thaliana[END_REF][START_REF] Choi | Arabidopsis homologs of components of the SWR1 complex regulate flowering and plant development[END_REF][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]). Yet, whether or not Arabidopsis CHR11/CHR17 and PIE/AtINO80 also participate in GA-related process remains currently unknown.

So far, only a few chromatin-remodeling factors have been reported in rice (Oryza sativa).

OsCHR4, a rice Chd-type chromatin-remodeling factor, was shown to be required for chloroplast development in the adaxial mesophyll [START_REF] Zhao | Molecular cloning and characterization of OsCHR4, a rice chromatin-remodeling factor required for early chloroplast development in adaxial mesophyll[END_REF]). CHR729, the rice homolog of PKL, was found to recognize methylated histone H3 lysine 4 (H3K4) and H3K27 and affect many aspects of plant development [START_REF] Hu | CHD3 protein recognizes and regulates methylated histone H3 lysines 4 and 27 over a subset of targets in the rice genome[END_REF]). Thus, it is important to characterize more rice chromatin-remodeling factors to uncover their biological functions.

In this study, we demonstrate that OsINO80-knockdown mutants display typical GAdeficient phenotypes and reduced levels of endogenous GAs. It is found that OsINO80 regulates GA biosynthesis by directly binding chromatin at the GA biosynthesis genes CPS1 and GA3ox2.

Our data further showed that OsINO80 interacts with H2A.Z and that loss of OsINO80 affects H2A.Z enrichment at these GA biosynthesis genes. Taken together, our study unravels important function of OsINO80 and provides a first link between chromatin-remodeling mechanism and GA biosynthesis regulatory pathway in the agronomic important crop rice.

RESULTS

Heterozygous osino80/+ mutant plants show a dwarf and late flowering phenotype

OsINO80 contains a long insertion embedded in the ATPase domain, which is the defining feature of Ino80-type distinct from other types of chromatin-remodeling factors (Figure S1A).

Both quantitative reverse transcription (qRT)-PCR analysis using RNA extracted from different rice tissues (Figure S1B) and histochemical GUS staining analysis using the reporter P OsINO80 ::GUS transgenic plants (Figure S1C) indicated that OsINO80 is ubiquitously expressed in various rice tissues.

To explore the role of OsINO80 in plant development, a T-DNA insertion mutant of OsINO80 (line PFG_1C-03165.R) was purchased from the database of T-DNA insertion mutant populations [START_REF] Yi | Utilization of T-DNA tagging lines in rice[END_REF]. This mutant was generated from the japonica-type cultivar 'Hwayoung' and has a T-DNA insertion in the first intron of the OsINO80 gene (Figure 1A).

We then performed backcrossing with the wild-type 'Hwayoung' for three generations to clean the mutant background. Using the hygromycin resistance gene embedded in the T-DNA insertion as the probe, we further performed Southern blotting to determine the T-DNA copy number in the mutant genome. When the genomic DNA of the heterozygous OsINO80 T-DNA insertion mutant (hereafter osino80/+) was digested with either the restriction enzyme EcoRV or HindIII, a single band was observed (Figure 1B), indicating that osino80/+ contains one T-DNA insertion.

Our PCR-based genotyping of 488 segregating plants from the self-pollinated osino80/+ failed to identify any homozygous mutant plants (Figure 1C). In contrast to the 1:2 segregation ratio as expected for an embryonic/zygotic lethal mutation, a segregation ratio of approximately 1:1 was observed for the wild-type 'Hwayoung' compared to osino80/+ ratio (Figure 1D), implying that not only embryonic lethal but also reduced gametophytic transmission has occurred in the mutant. Interestingly, the transcript level of OsINO80 was reduced to nearly half in osino80/+ (Figure 1E), and the heterozygous osino80/+ plants showed obvious morphological abnormalities as compared with the wild-type 'Hwayoung' (Figure 1F). The heterozygous osino80/+ plants are obviously dwarf at either the three-leaf (Figure S2A) or the heading developmental stage (Figure 1F). The panicle length of osino80/+ is shorter, and internode length is reduced as compared with that of wild-type (Figure 1G, Figure S2B and S2C). The dwarf phenotype of the heterozygotes osino80/+ may be caused by reduced cell elongation, because the cell length is obviously reduced in the second leaf sheath in the heterozygous mutant plants (Figure 1H and Figure S2D). Moreover, the osino80/+ mutants showed a late-flowering phenotype when grown side by side at two locations with different latitudes, Shanghai with long-day (LD) conditions, and Sanya with short-day (SD) conditions. Under both conditions, the average heading dates of osino80/+ were later than those of the wild-type plants. Only the data under LD conditions are shown in Figure 1I.

OsINO80 is involved in pollen germination and pollen tube elongation

To further investigate the function of OsINO80 in reproductive development, we introduced an enhanced yellow fluorescent protein (YFP)-tagged OsINO80 construct driven by the maize Ubiquitin promoter (P UBI ::OsINO80-YFP) into the osino80/+ plants. Using the PCR-based genotyping of progeny of self-pollinated transgenic plants (P UBI ::OsINO80-YFP/osino80/+), transgenic lines over-expressing OsINO80-YFP in the homozygous osino80-/-background (P UBI ::OsINO80-YFP/osino80-/-) (Figure S3A) were obtained, confirming that the homozygote lethality is indeed caused by a loss-of-function of OsINO80. Moreover, the transformation of P UBI ::OsINO80-YFP into homozygous osino80-/-partially rescued the physiological phenotypes shown in heterozygous osino80/+ (Figure S3B), indicating that YFP-tagged OsINO80 protein is functional in planta. We then performed the reciprocal cross-based analysis between osino80/+ and the wild-type 'Hwayoung' as shown in Table 1. The observed segregation ratio of F1 progeny was close to 1:1 according to the  2 -test when osino80/+ plant was used as a female. Conversely, when male osino80/+ plants served as pollen donors, the segregation ratio was 0.175:1, critically deviated from 1:1. It indicates that loss of OsINO80 mainly impairs male gametophyte development.

Because the osino80/+ mutant plants develop normal flowers with normal pistils and stamens, pollen development was analyzed using cytological observations. The viable pollen grains were stained dark by I 2 -KI solution, while the sterile grains were stained yellow or light red [START_REF] Chhun | Gibberellin regulates pollen viability and pollen tube growth in rice[END_REF]. As shown in Figure 2A, the pollen viability in osino80/+ is similar to that in wildtype 'Hwayoung'. No abnormities in the cell architecture of pollen grains between osino80/+ and wild-type 'Hwayoung' were found using 4',6-diamidino-2-phenylindole staining (Figure 2B). Next, we performed in vitro pollen germination assays. In wild-type 'Hwayoung', 93% of pollen grains germinated and elongated, while only 55% of those from osino80/+ plants germinated under the same conditions (Figure 2C and2D), suggesting that the defect of osino80/+ in reproductive development may be related to impaired pollen germination and pollen tube elongation.

RNA interference (RNAi) of OsINO80 plants display similar phenotypes as osino80/+

To further verify the role of OsINO80 in rice growth and development, we used an RNAi approach to knock down OsINO80 expression in rice. The nucleotides 2,038 to 2,239 and 4,849 to 5,234 of the OsINO80 transcript were selected to form the inverted repeats of a hairpin structure driven by the cauliflower mosaic virus 35S promoter, and the two constructs were named P 35S ::OsINO80-RNAi-1 and P 35S ::OsINO80-RNAi-2, respectively. P 35S ::OsINO80-RNAi-1 and P 35S ::OsINO80-RNAi-2 were transformed into the japonica-type cultivar Nipponbare to obtain the transgenic plants osino80Ri-1 and osino80Ri-2, respectively. After four generations, 10 independent osino80Ri-1 lines and 7 independent osino80Ri-2 lines were obtained. As expected, osino80Ri-1 and osino80Ri-2 plants displayed dwarf phenotypes similar to that of the T-DNA insertion mutant osino80/+. In correlation with their OsINO80 expression levels, osino80Ri-1 plants showed less severe phenotypes than those of osino80Ri-2 (Figure 3A and 3B). The lengths of the panicles and internodes of the osino80Ri-1 and osino80Ri-2 plants were shorter than those of the wild-type 'Nipponbare' plants at the mature stage (Figure S4A-S4C), and the 1,000-grain weight was also reduced in the RNAi mutants (Figure S4D). Moreover, we also observed a retardation in seed germination, a defect in pollen germination and a late-flowering phenotype in osino80Ri-2 plants (Figure S4E-S4G). Using osino80Ri-2 plants, which exhibited a more severe phenotype, we find that the decrease of plant height was probably caused by shorter cell length (Figure S4H andS4I).

OsINO80 modulates the transcription of a large number of genes involved in multiple processes, including GA-related genes

We then performed RNA sequencing (RNA-seq) to analyze global gene expression changes in osino80Ri-2 plants. By analyzing two independent biological replicates, 1,269 genes were down-regulated and 728 genes were up-regulated more than twofold in osino80Ri-2, compared with the wild-type rice (Table S1). Furthermore, a gene ontology (GO) analysis revealed that OsINO80 modulates many genes involved in a broad range of biological processes (Figure 3C and 3D). Both the down-and up-regulated genes in osino80Ri-2 enriched in functions related to stress response, including biotic stimuli, for example fungi as well as abiotic stimuli, such as oxidative stress, wounding, cold, and water deprivation (Figure 3C and 3D). Thus, our transcriptome analysis indicates that OsINO80 is required for normal expression of a large number of genes playing wide range of roles during plant growth and development.

OsINO80-knockdown mutants, derived from either T-DNA insertions or RNA interference, display typical GA deficient phenotypes such as delay of seed germination, plant body dwarfism, cell length reduction, late flowering, and reproductive defects. This prompts us to check the transcription levels of GA-related genes in osino80Ri-2 using the RNA-seq data.

Consistently, the transcription levels of the GA synthesis genes CPS1 and GA3ox2 were downregulated nearly 50% in osino80Ri-2 as compared to wild-type, albeit the transcription levels of GA signaling genes OsSLR1, OsGID1, and OsGID2 were not significantly changed (Table S2). Detail RNA-seq maps were shown for the down-regulation of OsINO80, CPS1 and GA3ox2 (Figure 3E-3G), and for the unchanged control gene OsGID1 (Figure 3H) in osino80Ri-2 compared with those in the wild-type plants.

OsINO80 is required for GA biosynthesis

To validate the mis-regulation of GA-related genes from the RNA-seq data, we analyzed the transcriptional levels of the GA-related genes by qRT-PCR assays in OsINO80-knockdown mutants and wild-type rice shoots. The examined genes include CPS1, KS1, KO2, KAO, GA20ox2, GA3ox2, and GA2ox1, which encode various enzymes involved in GA biosynthesis, and OsSLR1, OsGID1 and OsGID2, which are involved in GA signaling pathways. The transcriptional levels of the GA signaling genes OsSLR1, OsGID1, and OsGID2 were not obviously changed in osino80Ri-2 compared with those in the wild-type plants (Figure 4A).

Similar to the RNA-seq data, the genes CPS1 and GA3ox2, which are responsible for the synthesis of bioactive GA, were down-regulated in the osino80Ri-2 mutants compared to the wild-type plants, while the gene KAO, encoding an enzyme catalyzing the oxidation of entkaurenoic acid, was up-regulated (Figure 4A). The T-DNA insertion mutant osino80/+ resembled the OsINO80 RNAi mutants showing similar differential gene expression as analyzed using both rice shoots and anthers (Figure S5A and S5B), demonstrating that OsINO80 is required for GA biosynthesis through the modulation of genes encoding the key enzymes in the GA biosynthesis pathway. Moreover, the transformation of P UBI ::OsINO80-YFP into the homozygous osino80-/-rescued the GA-related gene mis-expression defects (Figure S5C).

We then examined the endogenous GAs in osino80Ri-2 and the wild-type plants. The levels of GA 12 , GA 53 , GA 19 , GA 20 and GA 1 were decreased in osino80Ri-2 compared with those in the wild-type rice (Figure 4B), validating a defect in GA biosynthesis in the OsINO80-knockdown mutants. To further verify the defects of osino80Ri-2 in GA biosynthesis, we performed an exogenous GA-treatment assay to check the responsiveness of osino80Ri-2. The plants were cultivated in Murashige and Skoog solid medium containing 0, 10 -10 , 10 -8 , 10 -6 , or 10 -4 M of the bioactive GA 3, and the length of the second leaf sheath was measured after 1 week. When the wild-type plants were treated with increasing concentrations of GA 3 , the length of the leaf sheath increased. For osino80Ri-2, the leaf sheath length also increased with treatments of increasing GA 3 concentrations, and its dwarf phenotype was fully rescued at 10 -4 M (Figure 4C). Similarly, the application of exogenous GA 3 also rescued the dwarf phenotype of the T-DNA insertion mutant osino80/+ at 10 -4 M (Figure S5D). All these data point to an important function of OsINO80 in regulation of the GA biosynthesis pathway.

OsINO80 directly targets to the GA biosynthesis genes CPS1 and GA3ox2

To analyze the function of OsINO80 protein in planta, we obtained transgenic plants overexpressing OsINO80-YFP driven by the maize Ubiquitin promoter (P UBI ::OsINO80-YFP) in the wild-type 'Nipponbare' background. The OsINO80-YFP transgenic plants showed an overexpression of OsINO80 (Figure S6A), but no disturbance to GA-related genes' expression (Figure S6B). By observing the root apical cells of P UBI ::OsINO80-YFP transgenic plants, we determined that, consistent with its chromatin-related function, the OsINO80-YFP protein was localized in the nucleus (Figure 5A).

Using the transgenic plants, we examined the enrichment of OsINO80 at the GA biosynthesis genes CPS1, GA3ox2, and KAO. The GA signaling gene OsGID1 served as a negative GAresponsive control. As shown in Figure 5B, transgenic plants overexpressed similar levels of OsINO80-YFP and YFP (P UBI ::YFP) [START_REF] Jin | MORF-RELATED GENE702, a Reader Protein of Trimethylated Histone H3 Lysine 4 and Histone H3 Lysine 36, Is Involved in Brassinosteroid-Regulated Growth and Flowering Time Control in Rice[END_REF]. ChIP analyses showed that OsINO80-YFP especially enriches at the 5' and 3' transcribed regions of the CPS1 and GA3ox2 genes, compared with the negative control YFP (Figure 5C and 5D), but not at those of KAO or OsGID1 (Figure S6C and S6D), indicating that CPS1 and GA3ox2 are the direct target genes of OsINO80, but KAO and OsGID1 are not. The up-regulation of KAO in OsINO80-knockdown mutants appears similar to that in some GA deficient mutants, such as gibberellin-deficient dwarf1 (gdd1) [START_REF] Li | Mutation of rice BC12/GDD1, which encodes a kinesin-like protein that binds to a GA biosynthesis gene promoter, leads to dwarfism with impaired cell elongation[END_REF], and the discrepancy between KAO expression and GA levels might be due to secondary effects and/or a post-transcriptional mechanism regulating the KAO protein level or function. Taken together, we conclude that OsINO80 directly binds to the GA biosynthesis genes CPS1 and GA3ox2, and maintains their expression level possibly through chromatin remodeling.

OsINO80 affects H2A.Z enrichment at GA biosynthesis genes

The next question is what will happen after OsINO80 enrichment at its target genes? H2A.Z is known as an H2A variant that performs essential functions in eukaryotes, and its dynamics are regulated and controlled by the ATP-dependent chromatin-remodeling complexes [START_REF] Sarma | Histone variants meet their match[END_REF][START_REF] Gerhold | INO80 and SWR complexes: relating structure to function in chromatin remodeling[END_REF][START_REF] Jarillo | H2A.Z mediates different aspects of chromatin function and modulates flowering responses in Arabidopsis[END_REF]. H2A.Z enriches at both active and silent genes, and may develop contrary transcription-related regulatory functions in a context-dependent manner [START_REF] Jarillo | H2A.Z mediates different aspects of chromatin function and modulates flowering responses in Arabidopsis[END_REF][START_REF] Subramanian | H2A.Z: a molecular rheostat for transcriptional control[END_REF]. The yeast INO80 acquires a histone-exchange activity by replacing nucleosomal H2A.Z/H2B with free H2A/H2B dimers [START_REF] Papamichos-Chronakis | Global regulation of H2A.Z localization by the INO80 chromatin-remodeling enzyme is essential for genome integrity[END_REF]. A recent study demonstrated that Arabidopsis INO80 interacts with H2A.Z and promotes key flowering genes' expression [START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF]. Therefore, we investigated the relationship between OsINO80 and H2A.Z in rice. We expressed a fragment of OsINO80 (299-567 AA) fused to a glutathione Stransferase (GST) tag. Using an in vitro pulldown assay, we found that GST-fused OsINO80, but not GST alone, preferentially interacted with H2A.Z (Figure 6A).

We find that the polyclonal antibodies against Arabidopsis H2A.Z [START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF] did not efficiently recognize rice H2A.Z (OsH2A.Z), probably because that several amino acids in the peptides of Arabidopsis H2A.Z used as the antigen are different from those in the rice H2A.Z. So we produced the specific antibody against rice H2A.Z using the peptides within HTA705 and HTA713 to verify the in planta interaction between OsINO80 and rice H2A.Z. As shown in Figure S7A, the OsH2A.Z antibody distinctively recognized the two typical rice H2A.Z proteins (HTA705 and 713) but not the H2A proteins (HTA702, 704 and 707) in western blot analysis. By using the rice mesophyll protoplasts overexpressing either the FLAG-tagged OsH2A (HTA702) or HA-tagged OsH2A.Z (HTA705) fusion proteins, only OsH2A.Z but not OsH2A could be recognized by the rice H2A.Z antibody (Figure S7B), further demonstrating the specificity of the antibody against OsH2A.Z. Using this rice H2A.Z antibody and the commercial H2A antibody, we performed a co-immunoprecipitation (Co-IP) experiment. The rice H2A.Z, but not H2A, was detected in the OsINO80-YFP immunoprecipitated fraction from the transgenic rice expressing OsINO80-YFP (Figure 6B), further confirming the physical interaction between OsINO80 and OsH2A.Z in vivo.

Using the rice H2A.Z antibody, ChIP-PCR analyses were performed in wild-type rice and OsINO80-knockdown mutants. We found that H2A.Z enriches near the 5' and 3' transcribed regions of CPS1 and GA3ox2 genes in the wild-type plants (Figure 6C and6D). Upon the knockdown of OsINO80, H2A.Z enrichments at the chromatin of GA3ox2 decreased obviously in both the T-DNA insertion mutant osino80/+ and RNAi mutant osino80Ri-2, similarly a decrease of H2A.Z enrichment at CPS1 was also observed (Figure 6C and6D). In contrast, the levels of H2A.Z at OsGID1 chromatin in the OsINO80-knockdown mutants were similar to those in the wild-type plants (Figure S8). When using the H3 antibody, H3 distribution patterns in CPS1 and GA3ox2 genes were observed similarly in wild-type and OsINO80-knockdown mutants (Figure 6C and 6D), indicating that OsINO80 does not affect the nucleosome positioning of its target genes. In OsINO80-knockdown mutants, the decreased H2A.Z but unchanged H3 distribution patterns at CPS1 and GA3ox2, combined with the down-regulation of these two genes, indicate that OsINO80 affects gene transcription in the GA biosynthesis pathway by altering H2A.Z deposition but not nucleosome occupancy.

DISCUSSION

Several Arabidopsis chromatin-remodeling factors, such as BRM, SWI3C, and PKL, have been reported to function in the GA pathway [START_REF] Henderson | PICKLE Acts throughout the Plant to Repress Expression of Embryonic Traits and May Play a Role in Gibberellin-Dependent Responses[END_REF][START_REF] Archacki | BRAHMA ATPase of the SWI/SNF Chromatin Remodeling Complex Acts as a Positive Regulator of Gibberellin-Mediated Responses in Arabidopsis[END_REF][START_REF] Zhang | The Chromatin-Remodeling Factor PICKLE Integrates Brassinosteroid and Gibberellin Signaling during Skotomorphogenic Growth in Arabidopsis[END_REF]). Here, we showed that both T-DNA insertion and RNAi mutants of OsINO80 displayed typical GA-deficient phenotypes, and the addition of exogenous GA 3 rescued the young seedling's dwarf phenotype of the mutants. Consistently, in the OsINO80-knockdown mutants, the GA biosynthesis genes CPS1 and GA3ox2 were down-regulated. CPS1 encodes an enzyme that catalyzes an early step in the GA biosynthetic pathway, producing GA 12 [START_REF] Sakamoto | An Overview of Gibberellin Metabolism Enzyme Genes and Their Related Mutants in Rice[END_REF].

The levels of endogenous GA 12 and the subsequent GAs, including GA 53 , GA 19 , GA 20 and bioactive GA 1 , were all decreased in the osino80Ri-2 mutant plants compared with those in the wild-type ones. Moreover, we found that OsINO80 protein directly binds to CPS1 and GA3ox2 and affects the H2A.Z deposition at these genes. Thus, our study establishes that OsINO80 is directly involved in GA biosynthesis, providing a first evidence in rice linking the phytohormone GA pathway with a chromatin-remodeling mechanism of regulation.

Chromatin-remodeling factors usually use ATP hydrolysis to facilitate histone octamer sliding, to remove or exchange histones, and to alter nucleosome positioning and structure, which leads to a modified chromatin structure during gene regulation [START_REF] Flaus | Mechanisms for ATP-dependent chromatin remodelling: farewell to the tuna-can octamer?[END_REF]. ChIP analyses using the histone H3 antibody showed that the OsINO80-knockdown mutants displayed similar patterns of nucleosome occupancy at CPS1 and GA3ox2 as the wildtype ones. Thus, OsINO80 affects the nucleosome composition of CPS1 and GA3ox2, without altering the nucleosome positioning. Several reports in Drosophila [START_REF] Rhee | Genome-wide structure and organization of eukaryotic pre-initiation complexes[END_REF][START_REF] Weber | Nucleosomes are context-specific, H2A.Z-modulated barriers to RNA polymerase[END_REF]) and in Arabidopsis [START_REF] Sura | Dual Role of the Histone Variant H2A.Z in Transcriptional Regulation of Stress-Response Genes[END_REF]) suggest that H2A.Z at the +1 nucleosome correlates with transcription activation. While a recent work proposes that H2A.Z in gene body associates with transcription repression in Arabidopsis [START_REF] Sura | Dual Role of the Histone Variant H2A.Z in Transcriptional Regulation of Stress-Response Genes[END_REF]). For the OsINO80 target genes CPS1 and GA3ox2, H2A.Z enrichments decrease both close to the +1 nucleosome and within 3' gene body in the OsINO80-knockdown mutants, so the downregulation of these two genes are probably due to the synergistic effects. In the future, to determine how OsINO80 protein is recruited to the target genes of CPS1 and GA3ox2 will help us to further understand the molecular mechanisms underlying OsINO80 regulation of the rice GA pathway.

Our previous work [START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF] and this study showed that AtINO80 and OsINO80 proteins share similar domain organization and H2A.Z specific binding activity, and they both affect plant height, flowering time and male gametophyte, but both genetic and molecular analyses indicate that AtINO80 and OsINO80 play also some different roles during plant growth and development. For example, when comparing the expressolog genes using the RNAseq data of osino80Ri-2 and the microarray data of atino80-5, we found that only 41 out of 767 down-regulated and 57 out of 938 up-regulated expressolog genes in atino80-5 overlap respectively with those in osino80Ri-2, suggesting the different effects of AtINO80 and OsINO80 on gene transcription (Figure S9). Moreover, in contrast with the viable and fertile phenotypes of the Arabidopsis mutant atino80-5 [START_REF] Zhang | The chromatin-remodeling factor AtINO80 plays crucial roles in genome stability maintenance and in plant development[END_REF], the OsINO80 loss-offunction mutant displayed more severe defects in reproductive development and viability in rice. We believe that future work in identifying the specific target genes of AtINO80 and OsINO80 will help us to understand their different biological roles.

We propose that OsINO80 is probably involved in GA-mediated pollen germination and pollen tube elongation, because GA plays important roles in pollen viability and pollen tube growth in rice [START_REF] Chhun | Gibberellin regulates pollen viability and pollen tube growth in rice[END_REF]). However, we cannot exclude the possibility that OsINO80 may directly modulate some genes involved in reproductive development. Likewise, we also cannot exclude the possibility that OsINO80 may directly regulate the genes responsible for flowering control. Because OsINO80 is widely expressed in different rice tissues, it will be interesting to investigate whether OsINO80 directly regulates more genes related to different biological processes at the chromatin level. RNA-seq analyses indicate that OsINO80 is required for the proper expression of many genes involved in diverse metabolic, biological, and cellular processes. Remarkably, many down-or up-regulated genes in OsINO80-knockdown plants enrich in functions related to stress responses and other phytohormones such as salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA) and growth hormone auxin. Thus, the transcriptome data not only highlighted the broad roles of OsINO80, but also provided direction for the further exploration of this chromatin-remodeling factor's functions.

MATERIALS AND METHODS

Plant material and growth conditions

The T-DNA insertion mutant line of OsINO80 (line PFG_1C-03165.R, generated from japonica-type cultivar 'Hwayoung') was obtained from the database of T-DNA insertion mutant populations [START_REF] Yi | Utilization of T-DNA tagging lines in rice[END_REF], and RNAi mutants have an O. sativa 'Nipponbare' background in this study. For phenotype analyses, plants were cultured in the paddy fields of Shanghai (LD photoperiod) and Sanya (SD photoperiod). Seedlings were grown in artificial growth chambers under LD conditions (30°C during 14-h light/28°C during 10-h dark) or SD conditions (30°C during 10-h light/28°C during 14-h dark) for molecular experiments.

Gene expression analysis

Two-week-old rice shoots were harvested for total RNA extraction using TRIzol reagent according to the manufacturer's instructions (Invitrogen; http://www.invitrogen.com). Reverse transcription was performed using Improm-II reverse transcriptase (Promega; http://www.promega.com) according to the standard protocol provided. Using gene-specific primers listed in Table S3, qPCR was performed. OsUbiquitin5 was used as the reference gene for data normalization.

GUS staining assay

The OsINO80 promoter fragment containing 2,000 bp nucleotides upstream of ATG was amplified by PCR using the primers listed in Table S3. The resulting DNA fragment replaced the cauliflower mosaic virus 35S promoter of pCAMBIA1301 to yield the construct P OsINO80 ::GUS, which was transformed into rice plants. Histochemical GUS assays using the transgenic plants of P OsINO80 ::GUS were performed as previously described [START_REF] Jefferson | GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants[END_REF].

Southern blot analysis

Genomic DNA was extracted and independently digested with two different enzymes, EcoRV and HindIII. The DNA fragments were separated on a 1.0% agarose gel and transferred onto a nylon membrane (GE Healthcare; http://www.gelifesciences.com). Then Southern blot analysis was performed with the DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche; http://www.roche-applied-science.com) using the hygromycin resistance gene as the probe.

Pollen-related assays

Pollen grains from the anthers of wild-type plants and OsINO80-knockdown mutants were analyzed by pollen I 2 -KI and 4',6-diamidino-2-phenylindole staining, and were germinated in vitro according to a previously described protocol [START_REF] Han | Rice Immature Pollen 1 (RIP1) is a regulator of late pollen development[END_REF][START_REF] Chhun | Gibberellin regulates pollen viability and pollen tube growth in rice[END_REF].

Transgene constructs and plant transformation

DNA fragments containing nucleotides 2,038 to 2,239 and 4,849 to 5,234 of the OsINO80 transcript were amplified as inverted repeats to construct a hairpin structure using the primers listed in Table S3. The hairpin structures were inserted into the vector pHB with a double cauliflower mosaic virus 35S promoter, obtaining P 35S ::OsINO80-RNAi-1 and P 35S ::OsINO80-RNAi-2 RNAi vectors for plant transformations. The full-length cDNA of OsINO80 was amplified by qRT-PCR using the primers listed in Table S3. The PCR product fused with the sequence encoding-enhanced YFP was then cloned into the vector pU1301 with the maize Ubiquitin promoter, obtaining P UBI ::OsINO80-YFP for plant transformations. The plant transformations were performed using the Agrobacterium tumefaciens (strain EHA105)mediated method described previously [START_REF] Sun | Rice jmjC domain-containing gene JMJ706 encodes H3K9 demethylase required for floral organ development[END_REF].

Bioinformatics analysis of RNA-seq data

The total RNA was extracted using TRIzol reagent (Invitrogen) from 2-week-old seedlings.

Library construction and sequencing were performed as described previously [START_REF] Liu | SET DOMAIN GROUP 708, a histone H3 lysine 36-specific methyltransferase, controls flowering time in rice (Oryza sativa)[END_REF].

Mapping information of RNA-seq data was shown in Table S4. The original paired-end reads were first trimmed using CUTADAPT v1.10 (Martin 2011). Then, TOPHAT2 v2.0.13 [START_REF] Kim | TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions[END_REF]) was used to align the adapter-removed raw reads to the complete reference genome of 'Nipponbare' (japonica) rice (http://rice.plantbiology.msu.edu). The normalized gene expression levels were calculated as fragments per kilobase of exon per million fragments mapped by CUFFLINKS v2.2.1 [START_REF] Trapnell | Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation[END_REF]. Next, the differentially expressed genes (DEGs) were identified using the CUFFDIFF program, which is a subpackage of CUFFLINKS.

The identification of the DEGs was performed at a threshold of more than twofold change and a P-value < 0.05.

For transcriptome comparison, the significant mis-regulated genes in Arabidopsis [START_REF] Noh | PIE1, an ISWI family gene, is required for FLC activation and floral repression in Arabidopsis[END_REF][START_REF] Fritsch | The INO80 protein controls homologous recombination in Arabidopsis thaliana[END_REF][START_REF] Choi | Arabidopsis homologs of components of the SWR1 complex regulate flowering and plant development[END_REF][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] were transformed to their homologous genes in rice firstly. Homologous genes data were downloaded from the web site (http://rice.plantbiology.msu.edu) and venn diagrams were generated with the 'Venn Diagram' packages in R software.

GO analysis

For the GO enrichment analysis, we uploaded the DEGs list to the online tool CARMO, which is a comprehensive annotation platform for the functional exploration of rice multi-omics data (http://bioinfo.sibs.ac.cn/carmo/Gene_Annotation.php) [START_REF] Wang | CARMO: a comprehensive annotation platform for functional exploration of rice multi-omics data[END_REF]. The significantly enriched biological processes among the DEGs (P < 0.05) were identified in OsINO80knockdown mutants.

Quantification of endogenous GAs

2-week-old seedlings (1 g) were frozen in liquid nitrogen, ground into fine powders and then 15 mL 80% (v/v) methanol extractions at 4°C for 12 h were performed. The endogenous GAs were analysed by the Greentek Corporation (http://www.greenswordcreation.com).

Exogenous GA 3 response assay

The seeds of wild-type rice and OsINO80-knockdown mutants were sterilized with 0.1% HgCl 2 for 15 min, washed four times using sterile distilled water, and finally planted on agar-solidified Murashige and Skoog medium containing 0, 10 -10 , 10 -8 , 10 -6 , and 10 -4 M bioactive GA 3 . After 1 week, the lengths of second leaf sheaths were measured.

ChIP assays

2-week-old rice shoots were used in ChIP assays as previously described [START_REF] Sui | H3K36 methylation is critical for brassinosteroid-regulated plant growth and development in rice[END_REF]. Antihistone H3 (ab1791) and anti-GFP (A-11122) were purchased from Abcam (http://www.abcam.com) and Invitrogen (http://www.invitrogen.com), respectively. The rabbit polyclonal antibodies specially recognized rice H2A.Z (OsH2A.Z) were generated against the mixed OsH2A.Z-specific peptides N-AGKGGKGLLAAKTTAAKAAADKDKDRKKAPVS-C (HTA705) and N-AGKGGKGLLAAKTTAAKSAEKDKGKKAPVS-C (HTA713) using a commercial service by Abmart (http://www.ab-mart.com.cn). Rice H2A (HTA702, 704, and 707) and H2A.Z (HTA705 and 713) cDNAs were amplified with primers listed in Table S3 and then cloned into the pGEX-4T-1 vector (Novagen; http://www.novagen.com). The purified rice H2A/H2A.Z proteins were used for detecting the recognition capacity of the OsH2A.Z antibody.

The enrichments of immunoprecipitated DNAs were determined by qPCR using a kit from Takara (http://www.takara-bio.com). Gene-specific primers are listed in Table S3

Protein binding assays

CDSs of histones H2A, H2B and H2A.Z were cloned into vector pET24a (Novagen; http://www.merckmillipore.com/). OsINO80 (299-567) cDNA was amplified using OsINO80 299-567 -F/R (Table S3) and cloned into the pGEX-4T-1 vector to construct GST-fused OsINO80 proteins. Pulldown experiments were performed according to a previously described protocol [START_REF] Bu | Regulation of arabidopsis flowering by the histone mark readers MRG1/2 via interaction with CONSTANS to modulate FT expression[END_REF]. The antibodies against GST (M20007) were purchased from Abmart (http://www.ab-mart.com.cn).

Protoplast isolation, transformation and immunoprecipitation (IP) assays

Protoplast isolation and transformation were performed using one to two weeks old rice shoots as described previously [START_REF] Choi | Trithorax group protein Oryza sativa Trithorax1 controls flowering time in rice via interaction with early heading date3[END_REF]. Rice H2A (HTA702) cDNA amplified using primers HTA702-PF/R was fused with FLAG cDNA and cloned into the pCAMBIA1300 vector driven by the cauliflower mosaic virus 35S promoter (P 35S ::FLAG-OsH2A). Rice H2A.Z (HTA705) cDNA amplified using primers HTA705-PF/R was fused with HA cDNA and cloned into the pU1301 vector with the maize Ubiquitin promoter (P UBI ::HA-OsH2A.Z). The constructs were transformed into protoplasts and the extracts were immunoprecipitated by anti-FLAG (F1084; Sigma; http://www.sigmaaldrich.com) or anti-HA (ab9110; Abcam; http://www.abcam.com) antibodies. The immunoprecipitated fraction was further detected by western blotting using anti-FLAG, anti-HA or anti-OsH2A.Z antibodies.

Co-immunoprecipitation (Co-IP) assays

2-week-old rice shoots over-expressing OsINO80-YFP (P UBI ::OsINO80-YFP) in wild-type 'Nipponbare' background were used to perform a Co-IP assay as described previously [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]). The proteins were extracted in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, 5 mM MgCl 2 , 0.1% NP-40, 1 mM EDTA, 2 mM DTT, anti-complete proteinase (Roche; http://www.roche-applied-science.com)). IP was performed using anti-GFP (M20004; Abmart; http://www.ab-mart.com.cn) in combination with pre-cleared magnetic protein A beads (Magna-ChIP; Millipore; http://www.merckmillipore.com) at 4°C for 3 h. After washing, the input and immunoprecipitated fractions were detected by western blotting using anti-GFP, anti-H2A (ab18255; Abcam; http://www.abcam.com) or anti- (C) Significant enriched biological processes identified by a gene ontology (GO) analysis of the down-regulated genes in the mutant osino80Ri-2 (P < 0.05).

(D) Significant enriched biological processes identified by a GO analysis of the up-regulated genes in osino80Ri-2 (P < 0.05).

(E-H) RNA-seq data showing the transcription levels of OsINO80 (E), CPS1 (F), GA3ox2 (G), and OsGID1 (H) in the wild-type 'Nipponbare' and osino80Ri-2.

  . The ChIP efficiency values are the ratios determined by taking a fixed aliquot of the DNA extracted from the immunoprecipitated samples and the ChIP input, and the fold change values in the figures are the normalized ChIP efficiency values using the reference gene OsUbiquitin5 as the internal standard. Error bars show standard deviation from three paralleled biological replicates.

Figure 1 .

 1 Figure 1. Characterization of T-DNA insertion mutant of Oryza sativa INO80. (A) Schematic representation of the OsINO80 gene structure and the T-DNA insertion in the line PFG_1C-03165.R. Black and gray boxes indicate for the coding sequences and untranslated regions, respectively. The right T-DNA border primer (RB) is located at the 3' end of the insertion. LP and RP are the left and right genomic primers, respectively, which were designed based on the OsINO80 sequence. (B) Southern blot analysis. Genomic DNA of the wild-type 'Hwayoung' and the mutant osino80/+ were digested using the restriction enzyme EcoRV or HindIII, and were analyzed through hybridizing using a hygromycin resistance gene as probe. (C) PCR amplification from genomic DNA of wild-type 'Hwayoung' and heterozygous osino80/+ plants using LP/RP and RB/RP primer pairs, respectively. (D) The segregation ratio of the progeny from self-pollinated heterozygous osino80/+ plants. (E) Relative expression levels of OsINO80. OsUbiquitin5 served as the internal control. Values shown are means ± standard deviations (SDs) of three independent replicates. Asterisks indicate statistically significant differences between osino80/+ and the wild-type 'Hwayoung' (P < 0.05). (F) Overall morphologies of the wild-type 'Hwayoung' and the mutant osino80/+. Bar = 15 cm. (G) Lengths of panicles and internodes of wild-type 'Hwayoung' and mutant osino80/+ plants. (H) Cell lengths in the second leaf sheaths of wild-type 'Hwayoung' and mutant osino80/+ plants. Asterisks indicate statistically significant differences between osino80/+ and the wildtype 'Hwayoung' (P < 0.05). (I) Heading time of the wild-type 'Hwayoung' and the mutant osino80/+ plants grown under LD (Shanghai) conditions. Values shown are means ± SDs (n = 30). Asterisks indicate statistically significant differences between osino80/+ and the wild-type 'Hwayoung' (P < 0.05).

Figure 2 .

 2 Figure 2. OsINO80 is required for pollen germination and pollen tube elongation. (A) Pollen grains stained dark by I 2 -KI solution. Bar = 100 m. (B) Pollen grains stained by 4',6-diamidino-2-phenylindole. V, vegetative nuclei; S, sperm

Figure 3 .

 3 Figure 3. RNA interference of OsINO80 impairs rice growth and disturbs gene expression in multiple processes. (A) Overall morphologies of the wild-type 'Nipponbare' and the mutant osino80Ri-1 and osino80Ri-2 plants. Bar = 15 cm. (B) Relative expression levels of OsINO80. OsUbiquitin5 served as the internal control. Values shown are means ± SDs of three independent replicates. Asterisks indicate statistically significant differences between the RNAi plants and the wild-type 'Nipponbare' (P < 0.05).

Figure 4 .

 4 Figure 4. OsINO80 is involved in regulation of the gibberellin (GA) biosynthesis pathway. (A) Relative expression levels of GA-related genes in the wild-type 'Nipponbare' and the mutant osino80Ri-2. OsUbiquitin5 served as the internal control, and the fold change relative to the wild-type level is shown. Values shown are means ± SDs from three independent replicates. Asterisks indicate statistically significant differences between osino80Ri-2 and the wild-type plants (P < 0.05).(B) The endogenous levels of GAs in the wild-type 'Nipponbare' and the mutant osino80Ri-2 plants. The numbers boxed are endogenous GAs levels in wild-type 'Nipponbare' (black) and osino80Ri-2 plants (orange). Data represent the means ±SD from three independent replicates in each line. Unit: ng•g-1 fresh weight; n.d., not detected; n.q., not quantified.(C) Effects of different concentrations of GA 3 on the wild-type 'Nipponbare' and the mutant

Figure 5 .

 5 Figure 5. OsINO80-YFP directly targets the GA biosynthesis genes CPS1 and GA3ox2. (A) Subcellular localization of OsINO80-YFP in rice root cells. Bar = 10 m. (B) Protein levels of YFP or OsINO80-YFP in the indicated genotypes detected by the GFP antibody. (C) Enrichment of OsINO80-YFP at CPS1 chromatin as analyzed by ChIP using GFP antibodies. Diagram representing CPS1 gene structure. Bars labeled 1-11 represent regions amplified by PCR. Error bars show the SDs from three biological replicates. (D) Enrichment of OsINO80-YFP at GA3ox2 chromatin as analyzed by ChIP using GFP antibodies. Diagram representing GA3ox2 gene structure. Bars labeled 1-7 represent regions amplified by PCR. Error bars show the SDs from three biological replicates.

Figure 6 .

 6 Figure 6. OsINO80 interacts with OsH2A.Z and affects OsH2A.Z enrichment at the GA biosynthesis genes CPS1 and GA3ox2. (A) Pulldown assays demonstrating the interaction of purified GST-tagged OsINO80 with H2A/H2B or H2A.Z/H2B recombinant proteins expressed in Escherichia coli. (B) Co-immunoprecipitation (Co-IP) detection of OsINO80 and OsH2A.Z interaction in planta. Total protein extracts from rice plants expressing OsINO80-YFP were immunoprecipitated with anti-GFP affinity beads and the resulting fractions were then analyzed by western blot using the antibodies against H2A or OsH2A.Z. (C) ChIP analysis using antibodies against OsH2A.Z or H3 at CPS1 chromatin in the indicated genotypes. Error bars show the SDs from three biological replicates. (D) ChIP analysis using antibodies against OsH2A.Z or H3 at GA3ox2 chromatin in the indicated genotypes. Error bars show the SDs from three biological replicates.

Table 1 .

 1 Reciprocal crosses between osino80/+ and wild-type 'Hwayoung'.

		Total	Number of	Number of
	Crosses	Number	osino80/+	Hwayoung	Observed	Expected	 2 -test Result
	(male × female)				Ratio	Ratio
		in F1	in F1	in F1
							 2 -value=28.070
	osino80/+ ×Hwayoung	114	17	97	0.175:1	1:1
							(p-value<0.01)
							 2 -value=1.110
	Hwayoung × osino80/+	73	32	41	0.780:1	1:1
							(p-value>0.05)
	Hwayoung ×
		94	0	94
	Hwayoung

ACKNOWLEDGEMENTS

This work was supported by the National Basic Research Program of China (973 Program, Grants no. 2012CB910500) and the National Natural Science Foundation of China (31570315, 91519308, and 31371304).

Data access

The RNA-seq data have been deposited in the NCBI SRA database (http://www.ncbi.nlm.nih.gov/sra) with the accession number SRP????.

OsH2A.Z antibodies.

AUTHOR CONTRIBUTIONS

SUPPORTING INFORMATION

Additional Supporting Information may be found online in the supporting information tab for this article: Venn diagrams show the number of genes mis-regulated.

Table S1. Genes down-or up-regulated by more than twofold in osino80Ri-2 compared with those in the wild-type plants.

Table S2. Transcription levels of OsINO80 and GA-related genes in the RNA-seq data of the wild-type and osino80Ri-2 plants.

Table S3. Primers used in the study.

Table S4. Mapping information of RNA-seq data.


Sylvain Guilley

Sumanta Chaudhuri

Laurent Sauvage

Tarik Graba

Jean-Luc Danger

Philippe Hoogvorst

Vinh-Nga Vong

Maxime Nassar

Florent Flament

Shall we trust WDDL?

Keywords: differential power analysis (DPA), side-channel attacks (SCA), secure logic style, dual-rail with precharge logic (DPL), wave dynamic differential logic (WDDL), early evaluation, timing unbalance

Security is not only a matter of cryptographic algorithms robustness but becomes also a question of securing their implementation. P. Kocher's Differential Power Analysis (DPA) is one of the many side-channel attacks that are more and more studied by the security community. Indeed, side-channel attacks (SCA) have proved to be very powerful on cryptographic algorithms such as DES and AES, customarily implemented in a wide variety of devices, ranging from smart-cards or ASICs to FPGAs. Among the proposed countermeasures, the "dual-rail with precharge logic" (DPL) aims at hiding information leaked by the circuit by making the power consumption independent of the calculation. However DPL logic could be subject to second order attacks exploiting timing difference between dual nets. In this article, we characterize by simulation, the vulnerability due to timing unbalance in the eight DES substitution boxes implemented in DPL WDDL style. The characterization results in a classification of the nodes according to their timing unbalance. Our results show that the timing unbalance is a major weakness of the WDDL logic, and that it could be used to retrieve the key using a DPA attack. This vulnerability has been experimentally observed on a full DES implementation using WDDL style for Altera Stratix EP1S25 FPGA.

Introduction

The principle of "dual-rail with precharge logic" (DPL) is to protect the implementation against side-channel attacks such as the Differential Power Analysis (DPA) [START_REF] Kocher | Differential Power Analysis[END_REF]. The DPL is a two-phase protocol composed of a precharge phase and a evaluation phase. Each signal is represented in dual-rail logic by its "true" and "false" parts, which are complementary. During the precharge phase, all signals are put in an initial state (assumed to be 00 in the sequel) to ensure that during the evaluation phase, the number of computations is fully predictable and constant whatever the inputs. In this article, we focus on the the "Wave Dynamic Differential Logic" (WDDL), a DPL based on standard cell flow, proposed by K. Tiri [START_REF] Tiri | A Logic Level Design Methodology for a Secure DPA Resistant ASIC or FPGA Implementation[END_REF].

However as described by D. Suzuki [START_REF] Suzuki | Security Evaluation of DPA Countermeasures Using Dual-Rail Pre-charge Logic Style[END_REF], DPL logic can still leak information because "true" and "false" parts can evaluate at different time according to the inputs. The root of this problem is the early evaluation. As a matter of fact, such difference in delay time will appear when early evaluation is combined with other unbalance factors, such as: difference of logical paths or unbalance of dual nets. Fig. 1b illustrates the principle of early evaluation for a 2-input AND gate and its dual 2-input OR gate, as represented on the Fig. 1a. We observe that, depending on the inputs value, the switching delay time differs because of the early evaluation in AND and OR gates. In this example, it is clear that the OR gate evaluates as soon as one of its inputs is set to '1', on an other hand, the AND gate must wait until each of its entries are set to '1', in order to switch its output. Of course, the opposite behavior would take place if the first available input would have been set to '0' (the AND gate would have evaluated immediately and the OR gate would have waited for every inputs to be set).

Early evaluation is then linked to the equations defining the circuit and exhibits the timing difference between signal a and b. In addition, for dual-rail logic, the difference of delay time could also be strengthened by two main factors:

1. The unbalance between the "true" and "false" nets, due to placement and routing differences, produces a timing difference between the true and the false net of a dual rail signal. In fact, if b t is slower than b f , the delay time ∆t 2 could increase as shown on Fig. 1c.

2. The unbalance of logical paths between inputs of a given logical gate, due to the fact that some inputs have to pass through more logical layers than others, leads to a timing difference between these input signals on each half path. In the same way, if a is slower than b, ∆t 2 could also increase significantly.

To summarize, the switching time of output s (s t ,s f ) depends on the inputs a (a t ,a f ) and b (b t ,b f ). As well as the switching time difference between s t and s f depends on the inputs. As a consequence, if for one signal, ∆t defined as ∆t .

= ∆t 1 -∆t 2 , can be detected by an instrument, the activity of the node could be monitored and this can lead to the secret key. In order to estimate if this vulnerability could be exploited on a real design, we have simulated the eight DES substitution boxes (S-Boxes) implemented in WDDL on an Altera Stratix FPGA and analyzed the switching timing delay for every node of the S-boxes. So far, difference in timing delay has only been studied on elementary designs, consisting of a single gate. Our objective is to assess whether or not this observation scales up to a full-fledged hardware encryption accelerator (in our case triple-DES on FPGA). To reach our goal, we first evaluate the vulnerability by simulation, and then effectively attack the physical implementation. The remainder of the paper is structured as follows: Sec. 2 presents the methods used to analyze the timing unbalance in dual-rail logic, Sec. 3 presents how WDDL could be successfully attacked based on a criterion defined in Sec. 2. Finally, the section 4 concludes this article. The appendix A provides with some precisions about the simulations realized in Sec. 2.

Timing Analysis of Differential Logic

We simulate the post-placed and routed compact substitution boxes (S-Boxes) designed specifically for FPGA as described in [START_REF] Sylvain Guilley | Place-and-Route Impact on the Security of DPL Designs in FPGAs[END_REF]. Each DES S-Box has a 6-bit input and a 4-bit output. The simulation environment provides the precharge and evaluation phases for all possible inputs. The main information extracted from the Value Change Dump (VCD) output file is the switching delay of each net during the precharge and evaluation phases. The switching delay is defined as the delay for one net to switch after the inputs are set. In our evaluations, we calculate for each node the absolute difference between the mean switching delay of the true net, and the mean switching delay of the false net. We call this difference ∆t. The nodes of the S-Box are then ordered in descending ∆t. The results are plotted for the eight S-boxes in Figure . 2.

According to Figure . 2, the most vulnerable S-Box seems to be box5 for two main reasons: ∆t is the highest and its decrease is the slowest.

The analysis yields a classification of the nodes according to their vulnerability, namely the ∆t. In order to exploit this information for real attack, the following methodology is applied:

1. First selection: find nodes where ∆t is higher than 1 ns (for experimental reasons: the timing difference should be visible with a sampling rate of 20 Gs/s and a bandwidth of 5 GHz).

2. Second selection: Among the nodes in the first selection, find the nodes where the dispersion of the switching delay do not overlap.

3. Third selection: Among the nodes in the second selection, select the ones having the smallest dispersion.

Figure . 3 presents the node selected for the eight S-Boxes of DES. It displays the repartition in time of the switching delay for the "true" and "false" nets. The node in box5 with nets wire e true and wire 1 f alse is chosen as the most vulnerable and will be used for a DPA attack on the DES implementation on our Altera Stratix FPGA (model EP1S25, in 130 nm technology). The separation between "true" and "false" evaluation dates is especially eloquent for S-Boxes 3, 4, 5 & 8.

Moreover, the simulation on the 2 6 = 64 possible inputs for the S-Boxes is also an easy way to extract information on the activity of each node. In fact, to apply a DPA on power traces collected on a real implementation, one should be able to define the selection function which depends on the node under attack. Based on the simulation, we can extract for each net, the list of inputs that make it switch, and therefore the selection function for the DPA.

Practical Test on DES WDDL Implemented in an Altera

Stratix EP1S25 FPGA

In order to validate the results obtained by simulation, we have performed a DPA attack on the node identified as the more vulnerable. We mention that attacks based on correlations with one single bit do work in practice on unprotected implementations. A differential trace after a single-bit correlation can be seen in Fig. 2 of [START_REF] Le | An overview of side channel analysis attacks[END_REF] or in Fig. 4.

As our objective here is only to validate that the timing difference for dual nets is visible, we make the correct assumption for the key and perform the DPA with the correct selection function. Figure . 5 shows the result of the DPA. We observe that the results match the expectation, e.g. that the DPA shows a spike that betrays the incriminated correlation.

Conclusion

Dual-rail with Precharge Logic (DPL) is a technique that ensures that upon execution, a circuit always toggles the same number of nodes during each clock cycle. This technique reduces significantly the information leakage on the power side channel (see left part of Figure . 5). With this technology, the attack based on the correlation between the key and the number of nodes toggled at a given clock cycle, is not possible anymore.

However some DPL technologies, such as WDDL, still leak information over the power side channel (see right part of Figure . 5). The switching timing difference between dual nets of some internal nodes is correlated to the data being computed. The power consumption profile of such chip is therefore exploitable to lead a side-channel attack.

We conclude that this correlation can be exploited for some nodes where the timing difference is significant enough. In addition, the analysis of the simulation leads to a classification of the nodes according to their vulnerability. For the first time, it has been proved that such correlation could be exploited on experimental traces. Finally, this article leads to the conclusion that the timing unbalance of dual nets should allow power consumption attacks. A Appendix: Details on Simulation and Timing Analysis The code snippet of Tab. 1 gives an example the VCD syntax. Tab. 2 presents an extract of the nodes classification for S-Box 5.

Figure 1 :

 1 Figure 1: Illustrations of dual-rail timing unbalance.

Figure 2 :

 2 Figure 2: ∆t decrease for the eight DES 6 → 4 S-Boxes.

S-Box 1 SFigure 3 :

 13 Figure 3: Switching delay of the most vulnerable nodes (true and false nets).

  drop across a 10Ω resistor [mV] DES round (R0 ⊕ R1)[START_REF] Kocher | Differential Power Analysis[END_REF]

Figure 4 :Figure 5 :

 45 Figure 4: Example of four mono-bit differential trace on SecMatV1 DES [2, §2.1].

Table 1 :

 1 Example of VCD file for a given gate.

	$scope module i_dut_1 $end
	$var wire 1 ! wire_a1_false $end
	$var wire 1 " wire_5e_true $end
	#0
	$dumpvars
	x"
	x!
	$end
	#1761
	0!
	#2605
	0"
	#51915
	1!
	#101348
	0!
	#151915
	1!
	#201348
	0!
	#252274
	1"
	#301464
	0"

Table 2 :

 2 Extract of nodes classification for S-Box 5.

	delta_t	wire/port name	activity	mean delay(ps)	std dev
	1336	wire_9a95_false	32	5224.00	738.12
		wire_656a_true	32	3888.00	318.50
	1152	wire_e_true	48	1696.00	116.12
		wire_1_false	16	2848.00	7.00
	1104	wire_9569_true	32	5096.00	469.32
		wire_6a96_false	32	3992.00	250.02
	1024	wire_3a5c_false	32	4416.00	269.61
		wire_c5a3_true	32	3392.00	222.74
	976	wire_69_false	32	4656.00	199.41
		wire_96_true	32	3680.00	197.14
	848	wire_9a_false	32	4064.00	812.79
		wire_65_true	32	3216.00	322.51
	...