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

Chao Li, Yuhao Liu, Wen-Hui Shen, Yu Yu, Aiwu Dong

► To cite this version:

Chao Li, Yuhao Liu, Wen-Hui Shen, Yu Yu, Aiwu Dong. Chromatin-remodeling factor OsINO80 is involved in regulation of gibberellin biosynthesis and is crucial for rice plant growth and development. Journal of Integrative Plant Biology, 2018, 60 (2), pp.144-159. 10.1111/jipb.12603. hal-02302871

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Submitted on 5 May 2023

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<figure><img src="image_1.png" /></figure>
Chromatin-remodeling factor OsINO80 is involved in regulation of gibberellin biosynthesis and is crucial for rice plant growth and development

Chao Li<fnref n="1" />, Yuhao Liu<fnref n="1" />, Wen-Hui Shen<fnref n="1" />,<fnref n="2" />, Yu Yu<fnref n="1" />*, and Aiwu Dong<fnref n="1" />,*

<footnote n="1">¹ State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China</footnote>

<footnote n="2">² Institut de Biologie Moléculaire des Plantes, UPR2357 CNRS, Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg Cédex, France</footnote>

<footnote>*Correspondences: aiwudong@fudan.edu.cn (Dr. Aiwu Dong is fully responsible for distributions of all materials associated with this article); yuy@fudan.edu.cn</footnote>

<footnote>Running title: OsINO80 promotes GA biosynthesis</footnote>
Abstract

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. OsINO80-
knockdown 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.

Keywords: rice; gibberellin, chromatin-remodeling factor, transcriptional regulation
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 (Hedden and Sponsel 2015). By searching for GA-
deficient 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) (Sakamoto 2004; Sun 2008).
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 (Ueguchi-Tanaka et al. 2007). 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 (Sakamoto 2004; Sun 2008).

GA biosynthesis and signaling pathways allow plants rapidly responding to environmental
changes, such as light, temperature, biotic and abiotic stresses (Yamaguchi 2008). Thus, GA-
related genes tend to be precisely regulated during plant growth and development (Yamaguchi
2008). 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 chromatin-
remodeling factors in eukaryotes, the Swi/Snf, Iswi, Chd, and Ino80 families (Clapier and
Cairns 2009). BRAHMA (BRM), the ATPase subunit of Swi/Snf-type chromatin-remodeling
complexes, is a positive regulator of GA-mediated responses in Arabidopsis (Archacki et al.
2013). 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 (Sarnowska et al. 2013). The Chd-type chromatin-
remodeling factor PICKLE (PKL) also interacts with DELLA proteins and functions in
Arabidopsis GA-dependent responses (Henderson 2004; Zhang et al. 2014). 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 (Huanca-Mamani et al. 2005; Li et al. 2012; Li et al. 2014), 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
(Noh and Amasino 2003; Fritsch et al. 2004; Choi et al. 2007; Rosa et al. 2013; Zhang et al.
2015). 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 (Zhao et al. 2012). 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 (Hu et al. 2012). 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 GA-deficient 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 (Yi and An 2013). 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 *Hind*III, 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*<sub>UBI</sub>::OsINO80-YFP) into the osino80/+ plants. Using the PCR-based
genotyping of progeny of self-pollinated transgenic plants (*P*<sub>UBI</sub>::OsINO80-YFP/osino80/+),
transgenic lines over-expressing OsINO80-YFP in the homozygous osino80-/- background
(*P*<sub>UBI</sub>::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<sub>UBI</sub>::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 χ<sup>2</sup>-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 I₂-KI solution, while the sterile grains were stained yellow or light red (Chhun et al. 2007). As shown in Figure 2A, the pollen viability in *osino80/+* is similar to that in wild-type '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 and 2D), 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*<sub>35S</sub>::*OsINO80-RNAi-1* and *P*<sub>35S</sub>::*OsINO80-RNAi-2*, respectively. *P*<sub>35S</sub>::*OsINO80-RNAi-1* and *P*<sub>35S</sub>::*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 and S4I).

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 down-regulated 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 ent-kaurenoic 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<sub>UBI::OsINO80-</sub>*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<sub>12</sub>, GA<sub>53</sub>, GA<sub>19</sub>, GA<sub>20</sub> and GA<sub>1</sub> 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<sup>-10</sup>, 10<sup>-8</sup>, 10<sup>-6</sup>, or 10<sup>-4</sup> M of the bioactive GA<sub>3</sub>, 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<sub>3</sub>, the length of the leaf sheath increased. For *osino80Ri-2*, the leaf sheath length also increased with treatments of increasing GA<sub>3</sub> concentrations, and its dwarf phenotype was fully rescued at 10<sup>-4</sup> M (Figure 4C). Similarly, the application of exogenous GA<sub>3</sub> also rescued the dwarf phenotype of the T-DNA insertion mutant *osino80/+* at 10<sup>-4</sup> 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 over-expressing *OsINO80-YFP* driven by the maize Ubiquitin promoter (*P<sub>UBI::OsINO80-YFP</sub>* ) 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<sub>UBI::OsINO80-YFP</sub>* 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 GA-responsive control. As shown in Figure 5B, transgenic plants overexpressed similar levels of OsINO80-YFP and YFP (*P<sub>UBI::YFP</sub>* ) (Jin et al. 2015). 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*) (Li et al. 2011), 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 (Sarma and Reinberg 2005; Gerhold and Gasser 2014; Jarillo and Pineiro 2015). H2A.Z enriches at both active and silent genes, and may develop contrary transcription-related regulatory functions in a context-dependent manner (Jarillo and Pineiro 2015; Subramanian et al. 2015). The yeast INO80 acquires a histone-exchange activity by replacing nucleosomal H2A.Z/H2B with free H2A/H2B dimers (Papamichos-Chronakis et al. 2011). A recent study demonstrated that Arabidopsis INO80 interacts with H2A.Z and promotes key flowering genes' expression (Zhang et al. 2015). 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 S-transferase (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 (Zhang et al. 2015) 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 and 6D). 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 and 6D). 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 (Henderson 2004; Archacki et al. 2013; Zhang et al.
2014). Here, we showed that both T-DNA insertion and RNAi mutants of OsINO80 displayed
typical GA-deficient phenotypes, and the addition of exogenous GA3 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 GA12 (Sakamoto 2004).
The levels of endogenous GA12 and the subsequent GAs, including GA53, GA19, GA20 and
bioactive GA1, 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 (Flaus and Owen-Hughes 2004). 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 wild-type ones. Thus, OsINO80 affects the nucleosome composition of CPS1 and GA3ox2, without altering the nucleosome positioning. Several reports in *Drosophila* (Rhee and Pugh 2012; Weber et al. 2014) and in *Arabidopsis* (Sura et al. 2017) 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* (Sura et al. 2017). 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 down-regulation 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 (Zhang et al. 2015) 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 RNA-seq 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* (Zhang et al. 2015), the *OsINO80* loss-of-function 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 (Chhun et al. 2007). 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 (Yi and An 2013), 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<sub>OsINO80::GUS</sub>*, which was transformed into rice plants. Histochemical GUS assays using the transgenic plants of *P<sub>OsINO80::GUS</sub>* were performed as previously described (Jefferson et al. 1987).

## 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 I₂-KI and 4',6-diamidino-2-phenylindole staining, and were germinated *in vitro* according to a previously described protocol (Han et al. 2006; Chhun et al. 2007).

## 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<sub>35S::OsINO80-RNAi-1</sub>* and *P<sub>35S::OsINO80-RNAi-2</sub>* 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<sub>UBI</sub>::OsINO80-YFP* for plant transformations. The plant
transformations were performed using the *Agrobacterium tumefaciens* (strain EHA105)-
mediated method described previously (Sun and Zhou 2008).

**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 (Liu et al. 2016).
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 (Kim et
al. 2013) 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 (Trapnell et al. 2010). 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 (Noh and
Amasino 2003; Fritsch et al. 2004; Choi et al. 2007; Rosa et al. 2013; Zhang et al. 2015) 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) (Wang et al. 2015). The significantly
enriched biological processes among the DEGs (*P* < 0.05) were identified in *OsINO80-
knockdown* 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<sub>3</sub> response assay

The seeds of wild-type rice and *OsINO80*-knockdown mutants were sterilized with 0.1% HgCl<sub>2</sub> for 15 min, washed four times using sterile distilled water, and finally planted on agar-solidified Murashige and Skoog medium containing 0, 10<sup>-10</sup>, 10<sup>-8</sup>, 10<sup>-6</sup>, and 10<sup>-4</sup> M bioactive GA<sub>3</sub>. 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 (Sui et al. 2012). Anti-histone 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. 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.
## 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<sub>299-567</sub>-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 (Bu et al. 2014). 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 (Choi et al. 2014). 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*<sub>35S</sub>::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*<sub>UBI</sub>::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*<sub>UBI</sub>::OsINO80-YFP) in wild-type 'Nipponbare' background were used to perform a Co-IP assay as described previously (Molitor et al. 2014). The proteins were extracted in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, 5 mM MgCl₂, 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-
OsH2A.Z antibodies.

## 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????.

## 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).

## AUTHOR CONTRIBUTIONS

A.D. conceived the project; C.L. performed most of the experiments; Y.L. performed the library construction and bioinformatics analyses of RNA-seq data; C.L., Y.L., Y.Y. and A.D. analyzed the data; C.L., WH.S., Y.Y. and A.D. wrote the paper.

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<table><caption>Table 1. Reciprocal crosses between *osino80/+* and wild-type ‘Hwayoung’.</caption><thead><tr><th>Crosses<br>(male × female)</th><th>Total<br>Number<br>in F1</th><th>Number of<br><i>osino80/+</i><br>in F1</th><th>Number of<br>Hwayoung<br>in F1</th><th>Observed<br>Ratio</th><th>Expected<br>Ratio</th><th>χ<sup>2</sup>-test Result</th></tr></thead><tbody><tr><td><i>osino80/+</i> × Hwayoung</td><td>114</td><td>17</td><td>97</td><td>0.175:1</td><td>1:1</td><td>χ<sup>2</sup>-value=28.070<br>(p-value&lt;0.01)</td></tr><tr><td>Hwayoung × <i>osino80/+</i></td><td>73</td><td>32</td><td>41</td><td>0.780:1</td><td>1:1</td><td>χ<sup>2</sup>-value=1.110<br>(p-value&gt;0.05)</td></tr><tr><td>Hwayoung ×<br>Hwayoung</td><td>94</td><td>0</td><td>94</td><td></td><td></td><td></td></tr></tbody></table>
FIGURE LEGENDS

**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 wild-type '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.** OsINO80 is required for pollen germination and pollen tube elongation.

(A) Pollen grains stained dark by I₂-KI solution. Bar = 100 µm.

(B) Pollen grains stained by 4',6-diamidino-2-phenylindole. V, vegetative nuclei; S, sperm
nuclei; Gp, germination pore. Bar = 50 µm.

(C) Pollen germination assays with pollen grains collected from the wild-type 'Hwayoung' and the *osino80/+* plants. Bar = 50 µm.

(D) Pollen germination rates in the wild-type 'Hwayoung' and the *osino80/+* mutant.

**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).

(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*.

**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 GA₃ on the wild-type 'Nipponbare' and the mutant
*osino80Ri-2*. The lengths of the second leaf sheaths were measured. Values are means ± SDs (n = 20). Asterisks indicate statistically significant differences between *osino80Ri-2* and the wild-type 'Nipponbare' (*P* < 0.05).

**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 µm.

(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.** 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.
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>Figure 1. Characterization of T-DNA insertion mutant of *Oryza sativa* INO80.</figcaption></figure>

(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 wild-type '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><img src="image_9.png" /><figcaption>Figure 2. OsINO80 is required for pollen germination and pollen tube elongation.</figcaption></figure>

(A) Pollen grains stained dark by I2-KI solution. Bar = 100 µm.

(B) Pollen grains stained by 4',6-diamidino-2-phenylindole. V, vegetative nuclei; S, sperm nuclei; Gp, germination pore.
Bar = 50 µm.

(C) Pollen germination assays with pollen grains collected from the wild-type 'Hwayoung' and the *osino80/+* plants.
Bar = 50 µm.

(D) Pollen germination rates in the wild-type 'Hwayoung' and the *osino80/+* mutant.
<figure><img src="image_10.png" /><figcaption>Figure 3. RNA interference of OsINO80 impairs rice growth and disturbs gene expression in multiple processes.</figcaption></figure>

(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).

(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*.
<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>Figure 4. OsINO80 is involved in regulation of the gibberellin (GA) biosynthesis pathway.</figcaption></figure>

(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 GA3 on the wild-type 'Nipponbare' and the mutant *osino80Ri-2*. The lengths of the second leaf sheaths were measured. Values are means ± SDs (n = 20). Asterisks indicate statistically significant differences between *osino80Ri-2* and the wild-type 'Nipponbare' (*P* < 0.05).
A

YFP

DIC

Merged

<figure><img src="image_14.png" /></figure>

B

OsINO80-YFP

YFP

KDa

<figure><img src="image_15.png" /></figure>

C

1000 bp

<figure><img src="image_16.png" /></figure>

D

250 bp

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /><figcaption>Figure 5. OsINO80-YFP directly targets the GA biosynthesis genes *CPS1* and *GA3ox2*.</figcaption></figure>

(A) Subcellular localization of OsINO80-YFP in rice root cells. Bar = 10 µm.

(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.
A

<figure><img src="image_20.png" /></figure>

B

<figure><img src="image_21.png" /></figure>

C

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

D

<figure><img src="image_24.png" /><figcaption>Figure 6. OsINO80 interacts with OsH2A.Z and affects OsH2A.Z enrichment at the GA biosynthesis genes *CPS1* and *GA3ox2*.</figcaption></figure>

(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.
SUPPORTING INFORMATION

Additional Supporting Information may be found online in the supporting information tab for
this article:

<figure><img src="image_25.png" /><figcaption>Figure S1. OsINO80 is broadly expressed throughout the plant.</figcaption></figure>

(A) Protein structure of OsINO80. Polygons indicate domains, as named in PROSITE (http://prosite.expasy.org/).

(B) Expression levels of *OsINO80* determined by quantitative RT-PCR in different tissues: leaf, root, and culm from seedlings, and leaf, flag leaf, culm and young panicles from mature plants. *OsUbiquitin5* served as an internal control. Values shown are means ± SDs of three independent replicates.

(C) Tissue expression pattern analyses of *OsINO80* by histochemical GUS staining in *P*<sub>*OsINO80::GUS*</sub> transgenic plants. Staining was performed in seedlings and panicles.
A

<figure><img src="image_26.png" /></figure>

B

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C

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D

<figure><img src="image_29.png" /><figcaption>**Figure S2.** Detailed morphologies of ‘Hwayoung’ and *osino80/+* plants.</figcaption></figure>

(A) Two-week-old seedlings of wild-type ‘Hwayoung’ (left) and *osino80/+* (right) plants. Bar = 3 cm.

(B) First panicles of wild-type ‘Hwayoung’ (left) and *osino80/+* (right) plants. Bar = 3 cm.

(C) Four uppermost internodes of wild-type ‘Hwayoung’ (left) and *osino80/+* (right) plants.
Bar = 3 cm.

(D) Cells of the second leaf sheaths in wild-type ‘Hwayoung’ and *osino80/+* plants. Bar = 20 µm. Arrows indicate the cell ends.
A

<figure><img src="image_30.png" /></figure>

B

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**Figure S3.** PCR identification and phenotype observation in *P<sub>UBI</sub>::OsINO80-YFP/osino80-/-*.

(A) PCR amplifications from genomic DNA of the indicated genotypes (top) using the indicated primer pairs (right).

(B) Overall morphologies of the wild-type ‘Hwayoung’, *osino80/+*, and *P<sub>UBI</sub>::OsINO80-YFP/osino80-/-*. Bar = 15 cm.
<figure><img src="image_32.png" /><figcaption>**Figure S4.** Detailed morphologies of *OsINO80*-knockdown plants by RNA interference.</figcaption></figure>

(A) First panicles of wild-type 'Nipponbare' (left), *osino80Ri-1* (middle), and *osino80Ri-2* (right) plants. Bar = 3 cm.
(B) Four uppermost internodes of 'Nipponbare' (left), *osino80Ri-1* (middle), and *osino80Ri-2* (right) plants. Bar = 3 cm.
(C) Lengths of panicles and internodes of wild-type 'Nipponbare', *osino80Ri-1*, and *osino80Ri-2* plants.
(D) One-thousand-grain weights of seeds from the wild-type 'Nipponbare', *osino80Ri-1*, and *osino80Ri-2* plants.
(E) Seed germination rates in the wild-type 'Nipponbare' and *osino80Ri-2* plants on Murashige and Skoog solid medium. Values shown are means ± SDs (n = 30).
(F) Pollen germination rates in the wild-type 'Nipponbare' and *osino80Ri-2* plants.
(G) Heading time of the wild-type 'Nipponbare' and *osino80Ri-2* plants grown under LD (Shanghai) conditions. Values shown are means ± SDs (n = 30).
(H) Cell lengths in the second leaf sheaths in wild-type 'Nipponbare' and *osino80Ri-2* plants.
(I) Cells of the second leaf sheaths in the wild-type 'Nipponbare' and *osino80Ri-2* plants.
(J) Arrows indicate the cell ends.
<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

**Figure S5.** The GA synthesis genes *CPS1* and *GA3ox2* are down-regulated in *osino80/+* and the exogenous GA₃ can rescue the dwarf phenotype of *osino80/+* young seedlings.

(A) Relative expression levels of GA-related genes in the indicated plants. *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 the *Osino80/+* and the wild-type (*P* < 0.05). (B) Relative expression levels of GA-related genes in the anthers of wild-type 'Hwayoung' and *osino80/+*. *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 the *osino80/+* and the wild-type (*P* < 0.05). (C) Relative expression levels of GA-related genes in the wild-type 'Hwayoung' and *PUBI::OsINO80-YFP/osino80/-* plants. *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. (D) Effects of different concentrations of gibberellic acid (GA₃) on the wild-type 'Hwayoung' and *osino80/+*. The lengths of the second leaf sheaths were measured. Values are means ± SDs (n = 20). Asterisks indicate statistically significant differences between *osino80/+* and the wild-type 'Hwayoung' (*P* < 0.05).
<figure><img src="image_37.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_38.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_39.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_40.png" /><figcaption>**Figure S6.** Gene expressions and ChIP analyses in *OsINO80-YFP* plants.</figcaption></figure>

(A,B) Relative expression levels of *OsINO80* (A) and GA-related genes (B) in the indicated plants. *OsUbiquitin5* served as the internal control. Values shown are means ± SDs from three independent replicates. Asterisks indicate statistically significant differences between *OsINO80-YFP* and the wild-type ‘Nipponbare’ (P < 0.05).

(C) Enrichment of OsINO80-YFP at *KAO* chromatin analyzed by ChIP analysis using GFP antibodies. Diagram representing *KAO* gene structure. Bars labeled 1–10 denote regions amplified by PCR. Error bars show standard deviations from three biological replicates.

(D) Enrichment of OsINO80-YFP at *OsGID1* chromatin analyzed by ChIP analysis using GFP antibodies. Diagram representing *OsGID1* gene structure. Bars labeled 1–7 denote regions amplified by PCR. Error bars show standard deviations from three biological replicates.
<figure><img src="image_41.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_42.png" /><figcaption>**Figure S7.** Specificity tests of the OsH2A.Z antibody *in vitro* and *in vivo*.</figcaption></figure>

(A) Purified GST-fused OsH2A or OsH2A.Z analyzed by western blotting using GST antibodies (top panel) or OsH2A.Z antibodies (bottom panel).

(B) Immunoprecipitated protein extracts from rice mesophyll protoplasts expressing FLAG-OsH2A and HA-OsH2A.Z analyzed by western blot using the antibodies against FLAG or HA.
<figure><img src="image_43.png" /><figcaption>**Figure S8.** ChIP analyses using antibodies against OsH2A.Z or H3 at *OsGID1* chromatin in indicated genotypes.</figcaption></figure>

Diagram representing the *OsGID1* gene structure. Bars labeled 1–7 denote regions amplified by PCR. Error bars show standard deviations from three biological replicates.
A

Down-regulated genes

<figure><img src="image_44.png" /></figure>

B

Up-regulated genes

<figure><img src="image_45.png" /></figure>

**Figure S9.** Comparison of the transcriptome profiles between rice *osino80Ri-2* and Arabidopsis *atino80-5* mutants.

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.