A natural allele of OsMS1 responds to temperature changes and confers thermosensitive genic male sterility

Wu, Lunying; Jing, Xiaohui; Zhang, Baolan; Chen, Shoujun; Xu, Ran; Duan, Penggen; Zou, Danni; Huang, Shengjian; Zhou, Tingbo; An, Chengcai; Luo, Yuehua; Li, Yunhai

doi:10.1038/s41467-022-29648-z

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Published: 19 April 2022

A natural allele of OsMS1 responds to temperature changes and confers thermosensitive genic male sterility

Nature Communications volume 13, Article number: 2055 (2022) Cite this article

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Abstract

Changes in ambient temperature influence crop fertility and production. Understanding of how crops sense and respond to temperature is thus crucial for sustainable agriculture. The thermosensitive genic male-sterile (TGMS) lines are widely used for hybrid rice breeding and also provide a good system to investigate the mechanisms underlying temperature sensing and responses in crops. Here, we show that OsMS1 is a histone binding protein, and its natural allele OsMS1^wenmin1 confers thermosensitive male sterility in rice. OsMS1 is primarily localized in nuclei, while OsMS1^wenmin1 is localized in nuclei and cytoplasm. Temperature regulates the abundances of OsMS1 and OsMS1^wenmin1 proteins. The high temperature causes more reduction of OsMS1^wenmin1 than OsMS1 in nuclei. OsMS1 associates with the transcription factor TDR to regulate expression of downstream genes in a temperature-dependent manner. Thus, our findings uncover a thermosensitive mechanism that could be useful for hybrid crop breeding.

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Introduction

As one of the most important cereal crops, rice (Oryza sativa L.) is a staple food for nearly half of the world’s population¹. Hybrid rice leads to a 10–20% grain yield increase compared with parental inbred lines^2,3,4, thereby contributing greatly to food security. The three-line system (the cytoplasmic male-sterile line [CMS], the maintainer line, and the restorer line) and two-line system (the thermosensitive genic male-sterile [TGMS] line/photoperiod-sensitive genic male-sterile [PGMS] line and the restorer line) are widely used for hybrid rice breeding^5,6. TGMS lines are male-sterile at restrictive (high) temperatures but male-fertile at permissive (low) temperatures. Therefore, the male-sterile line in two-line system also serves as the maintainer line, resulting in a dramatically decreased cost in seed production. Considering that most cultivars can restore the fertility of TGMS lines^5,7,8, two-line system utilizes broader genetic resources to generate hybrids than three-line system in hybrid rice breeding. Recently, TGMS lines have made tremendous contributions to heterosis breeding for increasing rice yield in China^2,5.

As the male gametophyte development has been identified as the most heat-vulnerable stage in flowering plants⁹, high temperature often causes male sterility. TGMS lines therefore provide a good system for studying the tissue-specific thermosensitive mechanisms. A few factors conferring TGMS phenotype have been identified in rice, including RNase Z^S1, long non-coding RNA, and receptor-like kinases, but temperature does not directly alter the properties (e.g., activity or protein abundance) of these factors^5,7,8.

Male reproductive development is a dynamic fine-tuning regulatory process involving the formation of male meiocytes, gametophytes, and gametes¹⁰. The male reproductive organ, the anther, forms male gametophytes via meiosis and mitosis within the flower, and releases mature pollen grain¹¹. Tapetum, the innermost anther wall layer, plays essential roles in supporting gametogenesis, and undergoes programmed cell death (PCD)-triggered degradation after the meiosis of microspore mother cells¹⁰. Thus, abnormal development and/or premature or delayed degradation of the tapetum results in male sterility. Several conserved receptor-like protein kinases and transcriptional regulators have been identified to be associated with tapetal cell development in rice¹⁰. Rice MULTIPLE SPOROCYTE 1 (MSP1) and MICROSPORELESS 2 (MIL2) encode receptor-like protein kinases and work coordinately to promote the correct progression of anther development^12,13. Rice MYB transcription factor encoded by GAMYB plays an important role in floral organ development and pollen development^14,15. Rice UDT1 (UNDEVELOPED TAPETUM 1)¹⁶, TDR (TAPETUM DEGENERATION RETARDATION)¹⁷, EAT1 (ETERNAL TAPETUM 1)¹⁸, and TIP2/bHLH142 (TDR INTERACTING PROTEIN 2)^19,20, encoding basic helix-loop-helix (bHLH) transcription factors, play key roles in maintaining tapetum development in rice. TIP2/bHLH142 acts downstream of UDT1 and GAMYB but upstream of TDR and EAT1 in pollen development^18,19,20. PTC1 (PERSISTENT TAPETAL CELL1)/OsMS1 (MALE STERILITY 1) encodes a transcriptional activator that regulates tapetal PCD and pollen exine formation in rice^21,22,23. However, how PTC1/OsMS1 is involved in the regulation of transcriptional activation remains unclear.

Here, we report that OsMS1 is a histone binding protein, and its natural allele OsMS1^wenmin1 from two indica TGMS lines (Tian1S and HengnongS-1) confers thermosensitive genic male sterility in rice. The abundances of OsMS1 and OsMS1^wenmin1 are temperature dependent. OsMS1 associates with the transcription factor TDR in nuclei to activate its downstream gene expression in a temperature-dependent manner. These findings define an important mechanism of thermosensitive genic male sterility control and open a way for hybrid crop breeding.

Results

The wenmin1 allele confers thermosensitive male sterility in both indica and japonica backgrounds

To dissect the temperature sensing mechanism, we collected multiple temperature-sensitive male-sterile lines used for two-line hybrid rice breeding. Among them, the indica TGMS line Tian1S showed male sterility under high temperature (restrictive temperature), but exhibited normal male fertility under low temperature (permissive temperature), irrespective of the day length (Supplementary Fig. 1a, b). Our genetic analysis revealed that the thermosensitive male-sterile phenotype was controlled by a single recessive locus (Supplementary Fig. 1c, Supplementary Table 2), hereafter named this locus wenmin1 (wenmin: temperature-sensitive in Chinese).

To explore whether the wenmin1 allele could also confer the thermosensitive male sterility in japonica varieties, we backcrossed Tian1S with a wide compatibility japonica cultivar ZhongJing1 (ZJ1) and generated the nearly isogenic line ZJ1-wenmin1, which contains the wenmin1 allele in ZJ1 background (Supplementary Fig. 2a, b). ZJ1-wenmin1 exhibited normal vegetative growth (Supplementary Fig. 2b), but was temperature-sensitive male sterile, irrespective of the day length (Supplementary Fig. 2c). To investigate the temperature range of wenmin1 in detail, we examined the pollen fertility of Tian1S and ZJ1-wenmin1 at day average temperatures (DAT) 22 °C, 23 °C, 25 °C, 27 °C, and 29 °C under the fixed photoperiods. Pollen fertility of Tian1S and ZJ1-wenmin1 was normal at 22 °C, whereas they exhibited a gradual abortion with increasing temperatures (Fig. 1a, b). Tian1S and ZJ1-wenmin1 showed no mature pollen grains at 27 °C and 25 °C, respectively. Together, these results indicate that the wenmin1 allele confers the temperature-sensitive male sterility in both indica and japonica cultivars at restrictive temperature and that the male sterility-fertility transition temperature of the wenmin1 allele depends on genetic backgrounds.

**Fig. 1: Phenotypes and identification of the *wenmin1* allele.**

We further examined anther development to characterize the cellular abnormalities of ZJ1-wenmin1. At restrictive temperature, the early development events of anthers and pollen grains of ZJ1-wenmin1 were similar to those of ZJ1, whereas ZJ1-wenmin1 tapetum appeared abnormally enlarged, and microspores became degenerated with few or no mature pollen grains compared with normal tapetal layer degradation and mature pollen grains formation of ZJ1 (Supplementary Fig. 2d).

Identification of the wenmin1 mutation

To identify the wenmin1 mutation, we conducted a map-based cloning by crossing Tian1S with ZJ1. Fine mapping of the wenmin1 mutation using 2102 F₂ individuals narrowed the candidate region to a 31.6 kb interval between markers RM24453 and A09-13 (Fig. 1c). Interestingly, this mapping region contains three genes, and one of them (LOC_Os09g27620) is the PTC1/OsMS1 gene^21,23. Notably, mutations in several PTC1/OsMS1 homologs including Arabidopsis thaliana MS1 (AtMS1)^24,25,26,27, barley MALE STERILITY 1 (HvMS1)²⁸, and maize MALE STERILITY 7 (ZmMS7)²⁹ resulted in the male sterility, although these mutations did not cause the temperature-sensitive male-sterile phenotype. Thus, LOC_Os09g27620 was the most promising candidate gene. To identify the causal mutation, we firstly investigated the expression of LOC_Os09g27620 in ZJ1 and ZJ1-wenmin1. We found that LOC_Os09g27620 is mainly expressed in stages 8 and 9 of anther development (Supplementary Fig. 3a), consistent with the public data (Supplementary Fig. 3b, c)³⁰. Similarly, OsMS1-GUS fusion proteins in gOsMS1:GUS transgenic plants were also detected in stages 8 and 9 of anther development (Fig. 1d). We observed that the transcript levels of LOC_Os09g27620 were similar in both ZJ1 and ZJ1-wenmin1 grown at restrictive temperature (Supplementary Fig. 3a), indicating that the mutation in ZJ1-wenmin1 does not affect the expression of LOC_Os09g27620. We then sequenced the LOC_Os09g27620 gene and found a nucleotide transition (T902C) in the second exon of LOC_Os09g27620 in Tian1S and ZJ1-wenmin1 compared with ZJ1 and indica cultivars 93-11 and Pei’Ai 64 (PA64) (Fig. 1c), which resulted in an amino acid change (Leu³⁰¹ to Pro, L301P) (Fig. 1e). These results suggest that T902C mutation in LOC_Os09g27620 might be responsible for the temperature-sensitive male-sterile phenotype of Tian1S and ZJ1-wenmin1. To confirm this, we conducted a genomic complementation assay. A genomic fragment of LOC_Os09g27620 (gOsMS1) from 93-11 was transformed into ZJ1-wenmin1. Transgenic plants (gOsMS1:ZJ1-wenmin1) showed normal fertility under restrictive temperature (Fig. 1f and Supplementary Fig. 4). Thus, these results demonstrate that the T902C mutation in LOC_Os09g27620/OsMS1 is responsible for the temperature-sensitive male sterile phenotype in Tian1S and ZJ1-wenmin1, and that the wenmin1 allele is a weak allele. Therefore, the wenmin1 allele was referred to as OsMS1^wenmin1 thereafter.

To explore whether other TGMS lines could contain the OsMS1^wenmin1 mutation, we sequenced the OsMS1 gene in multiple TGMS lines. The indica thermosensitive male sterile line HengnongS-1 (HNS-1) was bred from a cross progeny between wild rice and indica cultivars, and has been widely used in two-line hybrid rice breeding since the 1990s in China (China Rice Data Center, http://www.ricedata.cn). Interestingly, genomic DNA sequencing showed that HNS-1, but not other TGMS lines, harbors the same mutation as Tian1S and ZJ1-wenmin1 (Fig. 1c). Notably, the tms9-1 was also mapped to the OsMS1^wenmin1 region using an F₂ population from a cross between HNS-1 and Minghui63 (MH63)³¹. The OsMS1 was identified as the candidate gene for TMS9-1, although genomic complementation test has not been conducted³¹. Consistent with this, our allelic tests revealed that OsMS1^wenmin1 was allelic to tms9-1, but not to tms5 (Supplementary Table 5). We then analyzed the sequences of 4,726 rice genomes (http://ricevarmap.ncpgr.cn/v2) and found that none of these rice varieties contained the OsMS1^wenmin1 mutation, indicating that OsMS1^wenmin1 is a rare allele.

OsMS1 and its homolog genes are shared in monocot and dicot species (Supplementary Figs. 5 and 6), and the Leu³⁰¹ of OsMS1 is evolutionarily conserved (Supplementary Fig. 7), suggesting a promising common target to engineer TGMS lines in the plant kingdom.

OsMS1 is a histone binding protein

OsMS1 encodes a 683-amino acid protein that contains a putative nuclear localization motif (NLS) at the N-terminus, a LXXLL motif (where L is leucine and X is any amino acid) in the middle region³², and a plant homeodomain (PHD) in the C-terminus (Fig. 1e and Supplementary Fig. 5). In animals, the PHD fingers were proposed to play crucial roles in transcriptional regulation and were known to recognize a subset of post-translational histone modifications, such as histone H3 trimethylated at Lys4 (H3K4me3), unmethylated tail of histone H3 (H3K4me0), histone H3 trimethylated at Lys9 (H3K9me3), and histone H3 or H4 acetylated at various lysine residues (H3/H4Kac)^33,34,35. However, little is known about the function of tissue-specific expressed PHD proteins in plants³⁶. Interestingly, sequence alignment revealed that the PHD domain of OsMS1 is highly conserved in OsMS1 and its homologs (Supplementary Fig. 8a, b), but with a unique amino acid composition (Supplementary Fig. 8b, c), possessing more acidic amino acid that is different from the known PHD domains of human BPTF and PHF2 that specifically recognize H3K4me2/3, of human AIRE and BHC80 that bind H3K4me0, of human DPF3b that recognizes H3K14ac (Supplementary Fig. 8b, c). We therefore asked whether OsMS1 could bind histone tails through its PHD finger. To answer this, we tested several biotin-labeled histone H2A, H2B, H3, and H4 peptides and modified histone peptides bearing methylations and acetylations on different residues in peptide pull-down assays. Unexpectedly, FLAG-tagged full-length OsMS1 preferentially binds to H4 (lane 9), H4 acetylated at lysines 5, 8, 12, 16 (lanes 10–13), and H3 (lane 5) (Fig. 2a, upper panel). Meanwhile, OsMS1^wenmin1 also interacted with H4, acetylated H4, and H3 (Supplementary Fig. 9a), indicating that the mutation in OsMS1^wenmin1 does not affect its histone-binding ability. A glutathione S-transferase (GST) tagged C-terminal region of OsMS1 containing the PHD domain (GST-OsMS1C; amino acids 626–683) retains the histone-binding specificity of the full-length OsMS1 (Fig. 2a, middle panel), indicating that the PHD domain-bearing C-terminal region of OsMS1 is sufficient for binding these histone peptides. We further mutated the conserved C642, C645, H650, and C653, which are important for PHD domain structure, into Ala (Supplementary Fig. 8b). We found that the mutations (C642A, C645A, H650A, and C653A) within the PHD domain abolished the interactions between OsMS1 and histone peptides (Supplementary Fig. 9b), indicating that the PHD domain structure is crucial for the specific interaction. We confirmed the interaction between full-length OsMS1 and H4 in planta using a luciferase complementation imaging (LCI) assay and a bimolecular fluorescence complementation (BiFC) assay in N. benthamiana leaves (Fig. 2b, c). The association of OsMS1 with H4 in rice anthers is further demonstrated by the semi-in-vivo pull-down and in vivo co-immunoprecipitation assays (Fig. 2d, e). Together, these results indicate that OsMS1 is a histone binding protein.

The L301P mutation in OsMS1^wenmin1 affects its nucleus localization

Given the putative nuclear localization motif in OsMS1 (Fig. 1e and Supplementary Fig. 5) and the fact that the LXXLL motif was proposed to play a role in determining nuclear localization of Dax-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1)^37,38, we asked whether the L301P mutation in the conserved LXXLL motif of OsMS1 could influence its subcellular localization. We observed that the fluorescent signal of green fluorescent protein (GFP)-OsMS1 fusion protein was exclusively found in the nuclei in transgenic rice (Supplementary Fig. 10a), yeast (Fig. 3b and Supplementary Fig. 10b, upper panels), N. benthamiana (Supplementary Fig. 10c, upper panels), Arabidopsis (Supplementary Fig. 10d, upper panels), and rice protoplast (Supplementary Fig. 10e, upper panels), whereas GFP- OsMS1^wenmin1 is distributed in both the cytoplasm and nuclei (Fig. 3b, Supplementary Fig. 10b, lower panels, Supplementary Fig. 10c, middle and lower panels, Supplementary Fig. 10d, lower panels, Supplementary Fig. 10e, lower panels). To validate these results, we then investigated the localization of OsMS1 and OsMS1^wenmin1 using stable rice transgenic plants expressing OsMS1-FLAG and OsMS1^wenmin1-FLAG under native OsMS1 promoter. Consistent with the results in yeast, N. benthamiana, and Arabidopsis, we found that OsMS1 was exclusively detected in the nuclei, while OsMS1^wenmin1 was detected in both the cytoplasm and nuclei (Supplementary Fig. 10f). Together, these results indicate that the residue at position 301 (L301) is important for the nuclear localization of OsMS1, and also suggest that the L301P mutation affects the function of OsMS1, at least in part, by influencing its subcellular localization.

Temperature regulates the abundances of OsMS1 and OsMS1^wenmin1

Besides the histone binding domain, LXXLL motif also functions in transcriptional regulation in mammals^32,39. Notably, the L301P substitution in OsMS1^wenmin1 happens in the extremely conserved LXXLL motif (Fig. 1e and Supplementary Fig. 7). We therefore tested whether the L301P mutation in OsMS1 influences its transcriptional activation activity in yeast cell. Using BD-OsMS1 and BD-GFP-OsMS1 as baits and AD as prey, we found that OsMS1 and GFP-OsMS1 had the transcriptional activation activity in yeast cells (Fig. 3a). Furthermore, the PHD domain-bearing C-terminal region, but not the N-terminus, is necessary for the transcriptional activation of OsMS1 (Supplementary Fig. 11). Interestingly, we observed that yeast cells with BD-OsMS1^wenmin1 grew slowly at 22 °C, but had no obvious growth at 30 °C (Fig. 3a and Supplementary Fig. 12a), indicating a temperature-dependent transcriptional activation activity of OsMS1^wenmin1 in yeast cells. These results suggest that the L310P mutation in OsMS1^wenmin1 could suppress the transcriptional activation activity and/or decrease its protein abundance in a temperature-dependent manner. Consistent with this hypothesis, we observed that OsMS1 and OsMS1^wenmin1 protein abundance is sensitive to ambient temperatures in yeast cells (Fig. 3b). Compared with OsMS1, high temperature caused less OsMS1^wenmin1 accumulation in the nuclei (Fig. 3b and Supplementary Fig. 12b). Thus, it is possible that the L301P mutation in OsMS1^wenmin1 reduces its protein abundance in the nuclei, thereby decreasing the transcriptional activation activity in yeast cells, which is consistent with the fact that GFP-OsMS1 was detected in nuclei, while GFP-OsMS1^wenmin1 was distributed in both nuclei and cytoplasm (Fig. 3b and Supplementary Fig. 10b). However, we could not fully exclude the possibility that the L301P mutation may directly repress the transcriptional activation activity in yeast cells.

We next asked whether the abundances of OsMS1 and OsMS1^wenmin1 proteins are temperature-dependent in planta. We examined GFP-OsMS1 and GFP-OsMS1^wenmin1 protein levels using a transient expression assay in N. benthamiana. GFP signals of GFP-OsMS1 and GFP-OsMS1^wenmin1 were readily detectable at 22 °C, but became less abundant at 30 °C (Fig. 3c, d), although their transcript levels were similar (Supplementary Fig. 13a). Notably, GFP signals of GFP-OsMS1^wenmin1 in nuclei were obviously weaker than that of GFP-OsMS1 in nuclei (Fig. 3c, d and Supplementary Fig. 10c). We then quantified protein levels of OsMS1 and OsMS1^wenmin1 in response to ambient temperature changes using a dual-luciferase system (Fig. 3e). Surprisingly, we found that total luciferase activity of OsMS1-LUC and OsMS1^wenmin1-LUC at 22 °C was similar (Fig. 3f). Considering that the mutation in OsMS1^wenmin1 affects the distribution of OsMS1^wenmin1 in cytoplasm and nuclei, the luciferase activity of OsMS1^wenmin1-LUC in nuclei should be lower than that of OsMS1-LUC in the nuclei at 22 °C. The total luciferase activity of OsMS1^wenmin1-LUC was significantly lower than that of OsMS1-LUC at 30 °C (Fig. 3f). Importantly, the luciferase activity of both OsMS1-LUC and OsMS1^wenmin1-LUC was decreased at 30 °C compared with that at 22 °C (Fig. 3f), although OsMS1-LUC and OsMS1^wenmin1-LUC transcripts were not significantly affected by temperatures (Fig. 3g). We further examined OsMS1 and OsMS1^wenmin1 proteins using pOsMS1:OsMS1-GUS and pOsMS1:OsMS1^wenmin1-GUS transgenic rice plants grown at permissive or restrictive temperatures. Consistently, we found that GUS signals in both pOsMS1:OsMS1-GUS and pOsMS1:OsMS1^wenmin1-GUS transgenic lines were detectable at 22 °C, but became less apparent at 30 °C (Fig. 3h and Supplementary Fig. 14a). Notably, GUS signals in pOsMS1:OsMS1^wenmin1-GUS transgenic lines were barely detected at 30 °C (Fig. 3h, Supplementary Fig. 14a), although transcript levels of OsMS1 and OsMS1^wenmin1 in these transgenic lines were similar (Supplementary Fig. 13b–d). We then quantified protein levels of OsMS1-GUS and OsMS1^wenmin1-GUS using a GUS antibody. Consistently, we found that the abundances of OsMS1-GUS and OsMS1^wenmin1-GUS proteins were obviously lower at 30 °C than those at 22 °C, and that the abundance of OsMS1^wenmin1-GUS was barely detected at 30 °C (Fig. 3i, j and Supplementary Fig. 14b). To further examine the tissue expression pattern, we conducted transverse section analysis using stage 9 anthers of pOsMS1:OsMS1-GUS and pOsMS1:OsMS1^wenmin1-GUS transgenic plants grown at 22 °C. Consistent with the previous results²¹, we found that OsMS1-GUS was expressed in tapetum (Supplementary Fig. 14c). Notably, L301P mutation in OsMS1 did not affects its tapetum expression at 22 °C (Supplementary Fig. 14c). Taken together, these results indicate that ambient temperature affects the abundances of OsMS1 and OsMS1^wenmin1, and OsMS1^wenmin1 is more sensitive to temperature changes than OsMS1.

The interactions of OsMS1 and OsMS1^wenmin1 with TDR are temperature-dependent

Considering that OsMS1 has the transcriptional activation activity (Fig. 3a) and lacks a predicted DNA-binding domain (Fig. 1e and Supplementary Fig. 5), it is possible that OsMS1 could interact with transcription factors to regulate gene expression. To identify the potential OsMS1-interacting proteins, we used the N-terminal fragment of OsMS1 (OsMS1_1-625) as the bait to screen a yeast cDNA library derived from rice inflorescence, because the full-length OsMS1 had a self-activating activity. Interestingly, three independent clones corresponding to TDR were identified in this screen. We further confirmed the interaction of OsMS1_1-625 with the full-length TDR in yeast cells (Fig. 4a). We then observed the interaction between full-length OsMS1 and TDR in planta using a LCI assay in N. benthamiana leaves (Fig. 4b, c and Supplementary Fig. 15a, b). Furthermore, bimolecular fluorescence complementation (BiFC) assays in N. benthamiana leaves revealed that OsMS1 associates with TDR in the nuclei (Fig. 4d), where OsMS1 and TDR were colocalized (Supplementary Fig. 16). Consistently, co-immunoprecipitation assays showed that OsMS1 associates with TDR in transgenic rice (Supplementary Fig. 15c). The pull-down assay using in vitro translated GST-tagged OsMS1 and FLAG-tagged TDR further demonstrated a direct interaction between OsMS1 and TDR (Fig. 4e). Supporting this, OsMS1/PTC1 and TDR were spatio-temporally co-expressed (Supplementary Figs. 3, 16, and 17), and 40.3% of the altered genes in the tdr mutant had expression changes in the ptc1 mutant^17,21.

**Fig. 4: The interactions of OsMS1 and OsMS1^wenmin1 with TDR in a temperature-dependent manner.**

Considering that OsMS1^wenmin1 contributes to temperature-sensitive male sterility, we next asked whether OsMS1 and OsMS1^wenmin1 could interact with TDR in a temperature-dependent manner. Yeast cells cotransfacted AD with BD-OsMS1 but not BD-OsMS1^wenmin1 can grow in SD-4 plates (Supplementary Fig. 11). Using BD-OsMS1^wenmin1 as bait and AD-TDR as prey, yeast growth was observed on SD-4 plates incubated at 22 °C (Fig. 4a), albeit at a lower level. However, we did not observe yeast growth on SD-4 plates incubated at 26 °C or 30 °C (Fig. 4a). These results indicate that the temperature influences the abundance of OsMS1^wenmin1, thereby resulting in the reduced interaction between OsMS1^wenmin1 and TDR. Similarly, the temperature-dependent interaction between OsMS1^L304L305AA with mutations in LXXLL motif and TDR was also observed (Supplementary Fig. 18). We further used LCI assays to examine the interactions of OsMS1 and OsMS1^wenmin1 with TDR in planta. We found that the abundance of the OsMS1^wenmin1-TDR complex was temperature-dependent, and was also significantly lower than that of the OsMS1-TDR complex at both low and high temperatures (Fig. 4b, c and Supplementary Fig. 19a). Notably, we also observed that the interaction between OsMS1 and TDR was gradually decreased with increasing temperatures (Fig. 4b, c and Supplementary Fig. 19a), although the temperature did not obviously affect the interaction between OsMS1_1-625 and TDR in yeast cells (Fig. 4a). Furthermore, BiFC assays confirmed the temperature-dependent interactions of OsMS1 and OsMS1^wenmin1 with TDR in the nuclei (Fig. 4d and Supplementary Fig. 19b). Considering that high temperature reduced the levels of OsMS1 and OsMS1^wenmin1 proteins, and that the abundance of TDR proteins is not affected by the temperatures (Supplementary Fig. 20), it is possible that the temperature-dependent interactions of OsMS1 and OsMS1^wenmin1 with TDR are due to the temperature-dependent abundances of OsMS1 and OsMS1^wenmin1 in the nuclei. Supporting this idea, in vitro pull-down assay showed that OsMS1 and OsMS1^wenmin1 had similar ability to bind TDR (Fig. 4e).

The OsMS1-TDR complex activates EAT1 expression

To identify the downstream factors of the OsMS1-TDR complex in regulating male fertility, we examined the expression levels of several genes involved in male fertility regulation at different stages of anther development¹⁰. We found that expression levels of EAT1, CP1, and C6 in ZJ1-wenmin1 were significantly reduced in comparison to those in ZJ1 under restrictive temperature (Supplementary Fig. 21). Importantly, expression of EAT1, CP1, and C6 was also downregulated in tdr mutant^17,19, supporting that OsMS1 and TDR share common downstream targets. TDR has been known to bind E-box elements (CANNTG) to activate gene expression¹⁷. Although EAT1 has been proposed to act downstream of TDR to regulate tapetal development, and mutation in TDR resulted in the decreased expression of EAT1¹⁸, it is unclear whether TDR could directly bind the promoter of EAT1. To test this, we examined the promoter sequence of EAT1 and found putative TDR binding elements (CANNTG) (Fig. 5a). We subsequently performed electrophoretic mobility shift assays (EMSAs) to test whether TDR could bind the E-boxes in the EAT1 promoter. We expressed MBP-conjugated OsMS1 (MBP-OsMS1), TDR (MBP-TDR), or MBP in Escherichia coli. We found that MBP-TDR but not MBP-OsMS1 alone can bind directly to the promoter of EAT1 in vitro (Fig. 5b). Moreover, excess unlabeled probe can compete with the corresponding labeled probe and eliminate the observed shift (Fig. 5b), indicating the specificity of the interaction. These data support a direct interaction between TDR and the promoter of EAT1 via E-box elements. We further investigated the effects of OsMS1 and OsMS1^wenmin1 on the binding of TDR to the E-box elements in the EAT1 promoter. We found that OsMS1 enhances TDR’s binding to the EAT1 promoter (Fig. 5c). Notably, OsMS1^wenmin1 exhibited similar but a bit weaker ability than OsMS1 to enhance TDR’s binding to the EAT1 promoter (Fig. 5c).

**Fig. 5: OsMS1 enhances the binding of TDR to the *EAT1* promoter.**

Given the fact that OsMS1 and TDR are tapetum specific expressed genes (Supplementary Figs. 3)^17,21, we performed chromatin immunoprecipitation (ChIP)-qPCR assays in rice leaf protoplasts to investigate the effect of OsMS1 on the binding of TDR to the EAT1 promoter. We transiently expressed MYC-OsMS1 and MYC-TDR in rice protoplasts and detected the association of OsMS1 and TDR with the EAT1 promoter. ChIP-qPCR results showed that MYC-TDR, but not MYC-OsMS1 alone associated with the EAT1 promoter (Fig. 5d and Supplementary Fig. 22a). When MYC-OsMS1 was co-expressed with GFP-TDR, MYC-OsMS1 associated with the EAT1 promoter (Supplementary Fig. 22c, d), indicating that the association of OsMS1 with the EAT1 promoter depends on the TDR. In addition, we observed that OsMS1 enhanced the association of TDR with the EAT1 promoter, and OsMS1^wenmin1 exhibited similar but a bit weaker ability than OsMS1 to enhance the association of TDR with the EAT1 promoter in rice protoplasts (Fig. 5e and Supplementary Fig. 22b).

Finally, we performed ChIP-qPCR using pOsMS1:OsMS1-FLAG and pOsMS1:OsMS1^wenmin1-FLAG transgenic rice plants to test if OsMS1 and OsMS1^wenmin1 were enriched at the promoter region of EAT1 at different temperatures. We found that both OsMS1 and OsMS1^wenmin1 were enriched more at the EAT1 promoter at 22 °C than 30 °C (Fig. 5f, g). Notably, OsMS1^wenmin1 was barely enriched at the EAT1 promoter region at 30 °C compared with OsMS1 (Fig. 5f, g). We further examined the expression of EAT1 in ZJ1 and ZJ1-wenmin1 at different temperatures. We found that the expression of EAT1 in ZJ1 and ZJ1-wenmin1 were comparable at 22 °C, while expression of EAT1 was significantly decreased in ZJ1-wenmin1 at 30 °C compared with that in ZJ1 (Fig. 5h), indicating that OsMS1^wenmin1 influences the expression of EAT1 in a temperature-dependent manner.

Discussion

Male sterile lines are widely used for crop hybrid breeding, contributing greatly to food security. In rice, CMS lines and TGMS/PGMS lines are extensively utilized to explore hybrid vigor in three-line system and two-line system, respectively, which dramatically increase rice grain yield. As male reproduction is temperature-vulnerable in flowering plants⁹, rice TGMS lines give a good system to understand how plants sense and respond to changing ambient temperature. In this study, we discovered that the natural allele OsMS1^wenmin1 confers temperature-sensitive male sterility in rice, and OsMS1 is a PHD-finger protein that interacts with histones. Consistent with its role, OsMS1 is localized in nuclei. By contrast, OsMS1^wenmin1 is localized in both cytoplasm and nuclei, indicating that the mutation in OsMS1^wenmin1 influences its subcellular localization. We further found that temperatures regulate the abundances of OsMS1 and OsMS1^wenmin1, and OsMS1^wenmin1 is more sensitive to temperature changes than OsMS1. At permissive temperatures, the wild-type and the OsMS1^wenmin1 allele possess suitable levels of OsMS1 and OsMS1^wenmin1 proteins to keep normal pollen development, respectively, although the level of OsMS1^wenmin1 in the OsMS1^wenmin1 allele is lower in nuclei than that of OsMS1 in the wild type. Both OsMS1 and OsMS1^wenmin1 interact with the transcription factor TDR to activate the expression of downstream genes, thereby generating fertile pollens. At restrictive temperatures, the abundance of OsMS1 proteins was decreased, but there are still enough OsMS1 proteins in nuclei to interact with TDR to activate the expression of downstream genes, therefore producing fertile pollens. In contrast, the high temperatures much strongly reduce the OsMS1^wenmin1 protein levels, thus there are no enough OsMS1^wenmin1 proteins in nuclei to interact with TDR, resulting in a dramatic decrease of downstream gene expression and forming sterile pollens. Thus, our findings uncover an important thermosensitive mechanism by which OsMS1 interacts with the transcription factor to control male fertility in a temperature-dependent manner (Supplementary Fig. 23).

As sessile organisms, plant cells must be capable of adapting to various environmental signals, especially for ambient temperature^{40,41,42,43,44,45,46}. In plants, several proteins have been described to respond to ambient temperature^40,41,45,46. Factors that sense and respond to temperature have been proposed to exist in anywhere in the cell⁴⁷, and different tissues may also possess distinct mechanisms to detect temperature changes^43,44,47. We found that temperature controls the abundance of OsMS1, which is specifically expressed in anthers. The natural allele OsMS1^wenmin1 causes temperature-dependent male sterility. Thus, our results indicate that temperature-dependent abundance of OsMS1 in nuclei is crucial for tissue-specific response to temperature changes.

The LXXLL motif was originally reported to be a signature sequence that mediates the interactions of different proteins with nuclear receptors, and was proposed to be a defining feature of a new family of nuclear proteins in human⁴⁸. In animals, it was reported that LXXLL motifs influence nuclear localizations by interacting with nuclear proteins^32,38. For example, the LXXLL motif in the N-terminal repeat region of Dax-1 plays a crucial role in its nuclear localization through interacting with the nuclear protein Ad4BP/SF-1³⁸. Interestingly, the L301P mutation in the LXXLL motif of OsMS1^wenmin1 affects its subcellular localization. It will be interesting to investigate whether OsMS1 could interact with nuclear proteins to determine its nuclear localization and why the L301P mutation affects the nuclear localization of OsMS1^wenmin1 in the future.

LXXLL motifs are present in many transcription factors and cofactors, and involved in activating or repressing transcription³². The L301P mutation in the LXXLL motif of OsMS1^wenmin1 decreases its transcriptional activity (Fig. 3a, Supplementary Figs. 11 and 12a). Considering that the L301P mutation caused the reduced abundance of OsMS1^wenmin1 in a temperature-dependent manner, it is plausible that the changes in the subcellular localization and abundance of OsMS1^wenmin1 cause the decreased transcriptional activity. LXXLL motif was reported to mediate interactions between E proteins and p300/CBP histone acetyltransferases (HATs)³⁹. Notably, the L301P substitution in OsMS1^wenmin1 happens in the extremely conserved LXXLL motif (Fig. 1e and Supplementary Fig. 7). Considering that OsMS1 interacts with histones (Fig. 2a and Supplementary Fig. 9), we then conducted a mononuclease (MNase) digestion-qPCR assay to test whether OsMS1 could affect the chromatin status of the EAT1 promoter. As shown in Supplementary Fig. 24a, b, we found that the OsMS1^wenmin1 allele slightly influences the chromatin status of the EAT1 promoter. It seems plausible that OsMS1 might interact with histone modifiers (e.g., histone acetyltransferases) to influence the chromatin status and gene expression. In addition, a previous study showed that OsMS1 interacts with TIP2/bHLH142 in Y2H and BiFC assays²³, suggesting a possibility that OsMS1 may interact with multiple transcription factors to regulate gene expression.

Hengliangyouyihao bred from the indica HNS-1 with the OsMS1^wenmin1 allele and MH63 was once the most widely planted two-line hybrid rice in 1990s in China (http://www.ricedata.cn/). However, the relatively higher male sterility-fertility transition temperature of HNS-1 restricted its further application³¹. Rice breeders prefer to use TGMS lines that had 22–24 °C transition temperature in practical two-line hybrid rice breeding^7,49,50, because higher transition temperature usually causes self-pollination of TGMS lines during hybrid rice seed production under fluctuating environmental temperature conditions. We observed the transition temperatures of the OsMS1^wenmin1 allele depend on genetic backgrounds. The japonica ZJ1-wenmin1 confers temperature-sensitive genic male sterility with significantly decreased sterility-fertility transition temperature at ~23 °C (Fig. 1a), indicating that it has great potential as a better TGMS line for hybrid rice breeding. Considering that Leu³⁰¹ of OsMS1 is evolutionarily conserved in plant species, it opens a way to create TGMS lines by generating the L301P mutation in various rice cultivars and other crops using genome editing technology.

Methods

Plant materials and growth conditions

The rice parents, transgenic materials, and NILs used in this study were listed in Supplementary Table 1. All rice plants, including Tian1S (Oryza sativa L. ssp. indica), ZhongJing1 (Oryza sativa L. ssp. Japonica, ZJ1), ZJ1-wenmin1 (near-isogenic line of OsMS1^wenmin1 in the ZJ1 background), HengnongS-1 (Oryza sativa L. ssp. indica, HNS-1), AnnongS-1 (Oryza sativa L. ssp. indica, AnS-1), Zhu1S (Oryza sativa L. ssp. indica), Y58S (Oryza sativa L. ssp. indica), C815S (Oryza sativa L. ssp. indica), Pei’ai64S (Oryza sativa L. ssp. indica, PA64S) and Nongken58S (Oryza sativa L. ssp. Japonica, NK58S), Pei’ai64 (Oryza sativa L. ssp. indica, PA64), Zhonghua11 (Oryza sativa L. ssp. Japonica, ZH11), Nipponbare (Oryza sativa L. ssp. Japonica, NIP), 93-11 (Oryza sativa L. ssp. indica), gOsMS1:ZJ1-wenmin1 (genomic OsMS1 transgene in ZJ1-wenmin1 background), 35S:GFP-OsMS1 and 35S:GFP-OsMS1^wenmin1 (OsMS1 and OsMS1^wenmin1 overexpression in ZH11 background), and 35S: MYC-TDR (TDR overexpression in ZH11 background) transgenic lines were grown in open fields with 16.7 × 20 cm spacing.

Rice plants were planted in the open field until their inflorescence length reached ~0.5 cm, an indicator of anther development stage 3⁷, and then transplanted to the growth chamber under the indicated day average temperature (DAT). Different anther development stages were valued by flower and anther length⁵¹. The photoperiod was 12 h light and 12 h dark with 75% relative humidity unless specifically indicated.

For transverse section analysis of OsMS1-GUS and OsMS1^wenmin1-GUS in rice anther, pOsMS1:OsMS1-GUS and pOsMS1:OsMS1^wenmin1-GUS transgenic plants were grown at 22 °C or 30 °C in growth chamber.

Arabidopsis thaliana ecotype Col-0 was used as wild-type control. Arabidopsis plants were grown on soil in a growth chamber at 22 °C ± 2 °C under long-day photoperiod (16 h light/8 h dark) with light intensity of 120 μmol photons m⁻² s⁻¹. To grow Arabidopsis seedlings on MS medium, seeds were surface-sterilized for 10 min in 15% commercial bleach, washed thoroughly with sterile water, and plated on half-strength MS medium with 1% sucrose and 0.8% agar. Plants were stratified at 4 °C for 2 days in the dark and then transferred to a growth chamber at the indicated times at 22 °C ± 2 °C under long-day photoperiod (16 h light/8 h dark) with light intensity of 120 μmol photons m⁻² s⁻¹.

N. benthamiana plants were grown on soil in a growth chamber at 22 °C ± 2 °C under long-day photoperiod (16 h light/8 h dark) with light intensity of 120 μmol photons m⁻²s⁻¹. Five- to six-week-old N. benthamiana plants were used for transient expression assays.

Yeast strains and growth conditions

Saccharomyces cerevisiae strain AH109 was cultured in yeast extract peptone dextrose (YPD) media at the indicated temperatures. The presence of transgenes was confirmed by growth on SD/-Trp/-Leu plates (Clontech, Cat. No: 630317). The transformed yeasts were suspended in 0.9% NaCl to OD₆₀₀ = 1.0. Then, 3 μL of suspended yeast was spotted onto the indicated selection medium.

Bacterial strains and growth conditions

The Agrobacterium strain GV3101 was incubated overnight and resuspended in the activation buffer (10 mM MES, pH 5.6, 10 mM MgCl₂, and 150 µM acetosyringone). The desired Agrobacterium combinations were mixed and incubated at room temperature for at least 2 h by gentle rocking with the final concentration of each construct at OD₆₀₀ = 1. The activated Agrobacterium strains were then infiltrated into fully expanded N. benthamiana leaves through a 1 mL needleless syringe.

E. coli BL21(DE3) transformed with the indicated constructs were grown to a density OD₆₀₀ of 0.4–0.5 on LB supplemented with 100 µg/mL of ampicillin or 50 µg/mL Kanamycin. Protein expression was then induced by addition adding 0.1 mM IPTG and incubated 3 h at 37 °C with agitation.

Generation of ZJ1-wenmin1

A NIL for OsMS1^wenmin1 (ZJ1-wenmin1) was generated by backcrossing Tian1S with a wide compatibility japonica variety ZJ1 as the recurrent parent. The ZJ1-wenmin1 was selected from the BC₆F₂ population based on pollen fertility and marker-assisted selection.

Seed setting of Tian1S at permissive and restrictive temperatures

Tian1S and the wild-type control indica Pei’ai 64 (PA64) were planted in the open field in the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (40°22′N, 116°22′E) until their inflorescence length reached ~0.5 cm, and then transplanted to growth chamber for ~20 days at the indicated day average temperature (DAT) under photoperiod conditions of 12 h light and 12 h dark with 75% relative humidity. After full bolting, Tian1S and PA64 were replanted in the open field. Temperature profile set of the growth chamber was shown in Supplementary Table 3.

Pollen fertility analysis

For pollen fertility analysis in Fig. 1a, 100 anthers of 20 mature spikelets from four plants in heading date were sampled just before their blooming. All anthers were taken out and stained with 1% potassium iodide-iodine solution (I₂-KI). Pollen grains were released using a needle and debris was removed. More than 200 pollen grains were photographed in randomly picked microscopic fields under a Leica DM2500 microscope (Leica Microsystems) using a ×20 magnification objective to evaluate pollen fertility. Darkly stained and round pollen grains were defined as fertile, whereas unstained, lightly or incompletely stained, irregularly-shaped grains were defined as sterile. For detecting the pollen fertility of Tian1S at permissive temperature (22 °C) and restrictive temperature (30 °C) under photoperiod conditions of short day (10 h light and 14 h dark) and long day (14 h light and 10 h dark) in Supplementary Fig. 1b, temperature profile set of the growth chamber was shown in Supplementary Table 4. For examining pollen fertility in Fig. 1a, ZJ1, Tian1S, and ZJ1-wenmin1 were planted at the indicated different day average temperature (DAT) under photoperiod conditions of 12 h light and 12 h dark in growth chamber. For statistical analysis of Fig. 1a in Fig. 1b, ten repeats of pollen fertility experiments were conducted for ZJ1, Tian1S, and ZJ1-wenmin1, each with ten anthers being examined. For examining pollen fertility in Fig. 1f, ZJ1, ZJ1-wenmin1, and gOsMS1:ZJ1-wenmin1 lines (#1 and #2) were planted at natural high temperature (~30 °C, DAT).

Semi-thin section

For transverse section analysis of anthers in Supplementary Fig. 2d, spikelets at different developmental stages were collected and embedded. Briefly, anthers were collected and fixed in FAA fixative solution containing a mixture of 37% formaldehyde, 70% ethanol, and 100% acetic acid (1:18:1). Samples were further dehydrated through an ethanol series of 70%, 85%, 95%, and 100% (v/v). After embedding in Technovit 7100 resin (Hereaus-Kulzer, Wehrheim, Germany), transverse sections of 2 μm were cut using a RM2265 automated microtome (Leica, Germany), and stained with 0.25% toluidine blue O (Chroma Gesellshaft Shaud). The tissue sections were then imaged under a light microscope (Olympus BH2, Japan).

Map-based cloning of wenmin1

Tian1S is a thermosensitive male sterile indica variety. To identify the causal wenmin1 gene, we conducted a map-based cloning by crossing Tian1S with a wide compatibility japonica variety ZJ1. The wenmin1 was first delimited to a genomic interval between the SSR markers RM6235 and RM3600 on chromosome 9, and was then narrowed down to the markers RM24453 and A09-13, within a region covered by one BAC (AP005308). Three genes are located in this mapping region. Schematic structure of LOC_Os09g27620 showed that LOC_Os09g27620 has three exons and two introns. DNA sequencing showed that T902C mutation in LOC_Os09g27620 is responsible for the temperature-sensitive male sterility phenotype of Tian1S and ZJ1-wenmin1.

Notably, the OsMS1^wenmin1 mutation creates an Hpa II restriction site in the OsMS1 coding sequence. To distinguish OsMS1 and OsMS1^wenmin1, genomic DNA was isolated from rice seedlings by CTAB method. The resulting DNA was used in a 20 μL PCR reaction with the primers A09-37F and A09-37R. The PCR products were digested with Hpa II and then separated using 2% agarose gel electrophoresis. The Hpa II digested PCR products of OsMS1 and OsMS1^wenmin1 were 168 bp and 87 bp, respectively.

Allelism and functional complementation test

Hengnong S-1 (HNS-1), bred by the Hengyang Academy of Agricultural Sciences, was a widely used thermosensitive male sterile indica cultivar in the two-line hybrid breeding system in China (http://www.ricedata.cn/)⁵². To determine whether OsMS1^wenmin1 in Tian1S was allelic to the causal mutation in HNS-1, we performed an allelic non-complementation assay in the F₁ generation of a cross between Tian1S and HNS-1. To examine whether OsMS1^wenmin1 is allelic to tms5, we crossed Tian1S with Annong S-1 and Zhu1S, both of which are indica and carry tms5⁵. We also crossed Tian1S with 93-11 as a control. As shown in Supplementary Table 5, All F₁ heterozygous plants were grown in natural high-temperature conditions in open fields in Haikou (19°54′N, 110°33′E), Hainan Province when panicle development started in the summer. Male fertility was assayed as the seed setting rate, and plants with a seed setting rate lower than 10% were considered sterile.

For the functional complementation test, a fragment of 5.1 kb in length (designated gOsMS1), containing the entire OsMS1-coding region, along with 5′ and 3′ flanking regions was amplified from 93-11 genomic DNA using primers gOsMS1-F and gOsMS1-R, and cloned into the binary vector pMDC99. All constructs were sequencing-verified at TSINGKE Biological Technology (Beijing, China), and were individually transformed into Agrobacterium tumefaciens strain GV3101. gOsMS1 was used to transform the japonica TGMS line ZJ1-wenmin1. We chose ZJ1-wenmin1 for transformation because the indica variety Tian1S was difficult to transform. The presence of transgenes was determined by genomic PCR using oligonucleotide primers specific to the transgene constructs. We analyzed at least 10 independent lines.

Protein alignment and structure analysis

The protein sequences used for alignment are encoded by LOC_Os09g27620, LOC_Os09g27620, XP_006660674.1, XP_021309975.1, GRMZM5G890224, XP_004956970.1, BAK05033.1 and XP_003578202.1 for Nip (Nipponbare), 93-11, Ob (Oryza brachyantha), Sb (Sorghum bicolor), Zm (Zea may), Si (Setaria italic), Hv (Hordeum vulgare), and Bd (Brachypodium distachyon), respectively. Alignments of protein sequences were performed using GeneDoc software (http://nrbsc.org/gfx/genedoc). The nuclear localization signal was predicted using NLStradamus with default parameters (http://www.moseslab.csb.utoronto.ca/NLStradamus/)⁵³. The pre-loaded model was 2 state HMM dynamic, and the prediction cutoff value was 0.3. The plant homeodomain (PHD) was predicted using SMART (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1)⁵⁴.

Phylogenetic analysis of OsMS1 and its homologs

The protein sequence of OsMS1 was used to identify its homologs via BLAST search. A condensed phylogenetic tree was constructed with MEGA software (version 5.2)⁵⁵ using the Neighbor-Joining (NJ) method with the following parameters: Poisson model, Uniform rates, Complete deletion, and 1000-replicates Bootstrap. The homolog proteins are named according to species and their names or NCBI accession numbers. Bootstrap values (>50) are shown as percentages for each branch. Branch lengths are shown proportional to the amino acid variation rates. The genebank number for homolog proteins in Manihot esculenta, Jatropha curcas, Ricinus communis, Populus trichocarpa, Eucalyptus grandis, Medicago truncatula, Glycine max, Vigna angularis, Citrus clementina, Cucumis sativus, Brassica oleracea, Raphanus sativus, Camelina sativa, Arabidopsis lyrata, Solanum tuberosum, Nicotiana attenuata, Elaeis guineensis, Phoenix dactylifera, Sorghum bicolor, Setaria italica, Oryza brachyantha, Brachypodium distachyon, and Aegilops tauschii are OAY25374.1, KDP38178.1, XP_002521912.1, XP_002315838.1, XP_010043916.1, XP_003613773.1, XP_003518728.1, XP_017427127.1, XP_006448066.1, XP_004140423.1, XP_013610019.1, XP_018444384.1, XP_010493299.1, XP_020876299.1, XP_006355027.1, XP_019242345.1, XP_010917298.2, XP_017701237.1, XP_021309975.1, XP_004956970.1, XP_006660674.1, XP_003578202.1, and EMT13329.1, respectively.

Gene expression pattern analysis

Tissue-specific expression patterns of OsMS1 and TDR were obtained from Botany Array Resource (http://bar.utoronto.ca/efprice/cgi-bin/efpWeb.cgi)⁵⁶. The signal threshold was set to 1000 for all data extraction. The expression specificity of OsMS1 and TDR were also obtained from Rice Expression Profile Database (RiceXPro) (https://ricexpro.dna.affrc.go.jp/).

The quantitative real-time PCR analysis

Plant materials were ground into fine powders in liquid nitrogen and total RNA was isolated by RNAprep pure kit (TIANGEN, Cat. No: DP439-H) according to the manufacturer’s instructions. Total RNAs were treated with RNase-free DNase I (Sigma-Aldrich, Cat. No: 04716728001) according to the manufacturer’s protocol. Full-length cDNAs were synthesized using the FastQuant RT Super Mix kit (TIANGEN, Cat. No: KR108). qPCR reactions were performed on a Lightcycler 480 machine (Roche Applied Science, USA) using 2×SYBR Green supermix kit (BIO-RAD, Hercules, CA). To eliminate DNA contamination, qPCR-specific amplification was verified by a single band product in gel analysis. Data were calculated with the comparative cycle threshold (CT) method and normalized to the housekeeping gene OsACTIN1 in all qPCR experiments unless specifically indicated. Primers are summarized in Supplementary Data 1. For detecting OsMS1 and OsMS1^wenmin1 mRNA levels in Fig. 3g. Means of the LUC/RLUC mRNA ratios were normalized to OsMS1-LUC/RLUC at 22 °C.

Plasmid construction and plant transformation

All constructs used in this study were generated using an EZfusion kit (Genera, Cat. No: GR6086). To generate the plasmid 35S:GFP-OsMS1, the full-length open reading frame (ORF) of OsMS1 was amplified with the primers GFP-OsMS1-F and GFP-OsMS1-R from young panicles of indica variety 93-11 and cloned into pMDC43 vector. To generate the plasmid 35S:GFP-OsMS1^wenmin1, the ORF of OsMS1^wenmin1 was amplified with the primers GFP-OsMS1-F and GFP-OsMS1-R from young panicles of Tian1S and cloned into the pMDC43 vector. To generate the plasmid 35S:MYC-TDR, the ORF of TDR was amplified with the primers MYC-TDR-F and MYC-TDR-R from young panicles of ZH11 and cloned into the pCAMBIA1300-221-MYC vector. To generate the plasmid gOsMS1-GUS, the genomic fragment of OsMS1 was amplified with the primers gOsMS1-F and gOsMS1-R from young panicles of 93-11 and cloned into the pMDC164 vector. To generate the plasmid pOsMS1:OsMS1-GUS and pOsMS1:OsMS1^wenmin1-GUS, the full-length ORF of OsMS1 and OsMS1^wenmin1 was amplified and fused in frame with pOsMS1 and then cloned into the pMDC164 vector. 35S:GFP-OsMS1, 35S:GFP-OsMS1^wenmin1, 35S:MYC-TDR, pOsMS1:OsMS1-GUS, and pOsMS1:OsMS1^wenmin1-GUS were introduced into ZH11 via Agrobacterium-mediated transformation⁵⁷. 35S:GFP-OsMS1 and 35S:GFP-OsMS1^wenmin1 were also introduced into wild-type Arabidopsis Col-0 via Agrobacterium-mediated transformation using standard protocol⁵⁸. We analyzed at least 10 independent lines for each construct. Primers are summarized in Supplementary Data 1.

Peptide synthesis

Biotin-H2A (1-21), Biotin-H2B (1-21), Biotin-H3 (1-21), Biotin-H4 (1-21), Biotin-H4K5ac, Biotin-H4K8ac, Biotin-H4K12ac, and Biotin-H4K16ac peptides used in this study were biochemically synthesized by Bankpeptide Bio-Tech Co., Ltd (Hefei, China) with a purity >95%. Biotin-H3K4me3 (Cat. No: 81042), Biotin-H3K9ac (Cat. No: 81044), and Biotin-H3K14ac (Cat. No: 81001) were obtained from Active Motif. All peptides were dissolved in sterile pure water. Sequences of all peptides are summarized in Supplementary Table 6.

Histone pull-down assay

To generate the plasmid FLAG-OsMS1, the ORF of OsMS1 was amplified with the primers FLAG-OsMS1-F and FLAG-OsMS1-R and cloned into the pET-nT vector. To generate the plasmid FLAG-OsMS1^wenmin1, the ORF of OsMS1^wenmin1 was amplified with the primers FLAG-OsMS1-F and FLAG-OsMS1-R and cloned into the pET-nT vector. To generate the plasmid GST-OsMS1C, the ORF of OsMS1C was amplified with the primers GST-OsMS1C-F and GST-OsMS1C-R and cloned into the pGEX4T-1 vector. To generate the plasmid FLAG-OsMS1C with the mutations C642A, C645A, H650A, C653A, the synthesized mutated OsMS1C was amplified with the primers FLAG-OsMS1C-F and FLAG-OsMS1C-R and cloned into the pET-nT vector. To generate the plasmid FLAG-GST, the ORF of GST was amplified with the primers FLAG-GST-F and FLAG-GST-R and cloned into the pET-nT vector. To generate the plasmid FLAG-GFP, the ORF of GFP was amplified with the primers FLAG-GFP-F and FLAG-GFP-R and cloned into the pET-nT vector. The peptide pull-down assays were performed in a three hundred microliters system⁵⁹. Briefly, in vitro purified FLAG-OsMS1, FLAG-OsMS1^wenmin1, GST-OsMS1C (residues 626–683), FLAG-OsMS1C with the mutations C642A, C645A, H650A, C653A, FLAG-GFP, or FLAG-GST were stored in 50 mM Tris-HCl pH 7.4, 200 mM NaCl at −80 °C. Equal amounts (~5 μg) of purified FLAG-OsMS1, GST-OsMS1C, or FLAG-GST proteins in peptide-binding buffer (50 mM Tris-HCl 7.4, 200 mM NaCl, 0.05% NP-40) were precleaned with Streptavidin agarose beads (Sigma-Aldrich, Cat. No: 17-5113-01) to prevent nonspecific binding and then aliquoted equally into several parts. 1 μg of biotinylated histone peptides with different modifications was added to the individually aliquoted mixture and then incubated at 4 °C for half an hour with gentle rotation. Pull-down experiments were performed by adding 10 μL prewashed Streptavidin beads (Sigma-Aldrich, Cat. No: 17-5113-01) and incubated with rotation at 4 °C for half an hour. After washing with 3× 1 mL binding buffer for 1 min at 4 °C, the bound proteins were detected by immunoblotting using antibody to FLAG (Abmart, Cat. No: M20008, dilution 1:5000) or GST (Abmart, Cat. No: M20007, dilution 1:5000). Primers are summarized in Supplementary Data 1. For in vitro histone pull-down assay in Fig. 2a, the indicated proteins were pulled down by strepavidin beads and detected by western blots with antibody against FLAG (Abmart, Cat. No: M20008, dilution 1:5000) or GST (Abmart, Cat. No: M20007, dilution 1:5000). Empty streptavidin beads were used as negative binding control.

Semi-in vivo glutathione S-transferase pull-down assay

To generate the plasmid GST-OsMS1, the ORF of OsMS1 was amplified with the primers GST-OsMS1-F and GST-OsMS1-R and cloned into the pGEX4T-1 vector. Primers are summarized in Supplementary Data 1. To examine whether OsMS1 interacts with H4 in Fig. 2d, we conducted a semi-in vivo glutathione S-transferase (GST) pull-down assay using nuclear proteins of stage 9 rice anthers. In brief, purified GST-OsMS1 was incubated with nuclear proteins extracts from stage 9 anthers of rice young panicles. Proteins were pulled down by Glutathione Sepharose 4B (GE Healthcare, Cat. No: 45-000-139) and detected by western blots using anti-H4 antibody (Active motif, Cat. No: 61521, Clone: MABI 0400, dilution 1:5000) or anti-GST antibody (Abmart, Cat. No: M20007, dilution 1:5000). GST was used as a negative control.

Co-immunoprecipitation assay

To confirm the interaction between OsMS1 and H4 in Fig. 2e, stage 9 anthers of rice young panicles of 35S:OsMS1 transgenic rice was immunoprecipitated with GFP-Trap^®_A agarose beads (Chromotek, Cat. No: gta-20), then endogenous histone H4 was detected using antibody against H4 (Active Motif, Cat. No: 61521, Clone: MABI 0400, dilution, 1:5000).

Homozygous transgenic plants expressing 35S:GFP and 35S:GFP-OsMS1 were crossed with transgenic plants expressing 35S:MYC-TDR, the double transgene plants of 35S:GFP/35S:MYC-TDR and 35S:GFP-OsMS1/35S:MYC-TDR were determined by PCR, and used to confirm the interaction between OsMS1 and TDR. Briefly, about 0.5 g young panicles with stage 9 anthers were grinded into powder in liquid nitrogen. Notably, all the subsequent steps were carried out in a 4 °C cold room or on ice. Briefly, about two volumes of protein extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 5% glycerol, 1 mM EDTA, 1× Roche protease inhibitor cocktail) were added to each sample to homogenize the powder. The resuspended samples were centrifuged at 13,523 × g for 10 min and the supernatant was subsequently transferred to new 1.5 mL tubes. GFP-Trap^®_A agarose (Chromotek, Cat. No: gta-20) was then added to the tubes and incubated at 4 °C for 30 min with gentle rocking. The beads were collected by centrifugation and washed at least three times with the wash buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% Triton X-100, 5% glycerol, 1 mM EDTA, 1× Roche protease inhibitor cocktail). Finally, the bound proteins were eluted by heating the beads in a 1×SDS loading buffer at 95 °C for 5 min. The eluted proteins were separated in a 10% (w/v) SDS-polyacrylamide gel, and probed with an anti-GFP antibody (Abmart, Cat. No: M20004, dilution, 1:5000), an anti-MYC antibody (Abmart, Cat. No: M20002, dilution, 1:5000) or anti-H4 antibody (Active Motif, Cat. No: 61521, Clone: MABI 0400, dilution, 1:5000).

Transcription activation assay

Transcription activation assay was performed using Matchmaker GAL4 Two-Hybrid System 3 (Clontech, USA) following the manufacturer’s instructions. The full-length and a series of truncations or mutations of OsMS1 were amplified and fused with the DNA-binding domain (BD) in vector pGBKT7 (Clontech, USA). All the resulting constructs were sequencing-verified and cotransformed with pGADT7 (Clontech, USA) into Saccharomyces cerevisiae strain AH109. The presence of transgenes was confirmed by growth on SD/-Trp/-Leu plates (Clontech, Cat. No: 630317). The transformed yeasts were suspended in 0.9% NaCl at OD₆₀₀ = 1.0. Then, 3 μL of suspended yeast was spread into SD/-Trp/-Leu/-His (Clontech, Cat. No: 630319) + 2 mM 3-aminotriazole (Sigma-Aldrich, Cat. No: A8056) or SD/-Trp/-Leu/-His/-Ade (Clontech, Cat. No: 630323) plates. Yeast growth was observed after incubation at the indicated temperatures. For detecting the transcriptional activation activity in Fig. 3a, yeast cells were spotted on SD-2 or SD-3 plus 2 mM 3-aminotriazole (3-AT) plates and grown at low (22 °C) or high (30 °C) temperatures for 4d. For quantifying the transactivation activities in Fig. 3a, we conducted yeast liquid assays. Equal numbers of diploid yeast cells were cultured in the SD-2 or SD-3 liquid medium at 22 °C (OD₆₀₀ = 0.05) and 30 °C (OD₆₀₀ = 0.01) for the indicated time. Relative yeast growth was shown as (OD₆₀₀ in SD-3)/(OD₆₀₀ in SD-2).

Yeast two-hybrid assay

Yeast two-hybrids were performed using Matchmaker GAL4 Two-Hybrid System 3 following the manufacturer’s instructions. As the full-length OsMS1 autoactivated the reporter gene when fused with BD, we used the N-terminal fragment of OsMS1 (referred to as OsMS1_1-625, amino acids 1-625) as bait to screen a cDNA library derived from rice inflorescence.

To check for the interaction between OsMS1_1-625, OsMS1^wenmin1, OsMS1^L304L305AA, and TDR, the coding sequence of OsMS1^wenmin1, OsMS1^L304L305AA, and OsMS1_1-625 were fused with the BD in pGBKT7 (Clontech, Cat. No: 630443), and the coding sequence of TDR was fused with the DNA-activation domain (AD) in pGADT7 (Clontech, Cat. No: 630442). Constructs for testing interactions were cotransformed into yeast strain AH109. The presence of transgenes was confirmed by growth on SD/-Trp/-Leu plates (Clontech, Cat. No: 630317). The transformed yeasts were suspended in 0.9% NaCl to OD = 1.0. Then, 3 μL of suspended yeast was spotted onto a stringent selection medium SD/-Trp/-Leu/-His/-Ade (Clontech, Cat. No: 630323). Interactions were observed after incubation at the indicated temperatures. For detecting the temperature-dependent association of OsMS1^wenmin1 and TDR in Fig. 4a, yeast cells (OD₆₀₀ = 1) were spotted on the SD-4 plates and grown at 22 °C, 26 °C, and 30 °C for 96 h.

To quantify the temperature-dependent association of OsMS1^wenmin1 and OsMS1^L304L305AA with TDR in yeast in Supplementary Fig. 18a, equal amounts of yeast diploid cells were cultured in 10 mL liquid SD-4 (Clontech, Cat. No: 630322) medium at 22°C, 26°C, and 30°C for about 96 h until yeast cells coexpressing OsMS1_1-625-BD and TDR-AD were grown to an OD₆₀₀ of ~2.0. Yeast growth were shown as OD₆₀₀ and normalized to yeast cells coexpressing OsMS1_1-625-BD and TDR-AD.

Luciferase complementation imaging assay

For confirming OsMS1-interacting proteins, coding sequences of OsMS1 were cloned into vector pCAMBIA1300-35S-nLUC-RBS⁶⁰ using primers OsMS1-Nluc-F and OsMS1-Nluc-R. The full-length ORF of TDR was cloned into the vector pCAMBIA1300-35S-cLUC-RBS using primers Cluc-TDR-F and Cluc-TDR-R. All the constructs were introduced into Agrobacterium tumefaciens strain GV3101. Primers are summarized in Supplementary Data 1. The LCI assay was carried out in N. benthamiana leaves^61,62. In brief, Agrobacterium strains GV3101 with individual expression constructs were incubated overnight and resuspended in the activation buffer (10 mM MES, pH 5.6, 10 mM MgCl₂ and 150 µM acetosyringone). The desired Agrobacterium combinations were mixed and incubated at room temperature for at least 2 h by gentle rocking with the final concentration of each construct at OD₆₀₀ = 1. The activated Agrobacterium strains were then infiltrated into fully expanded N. benthamiana leaves through a 1 mL needleless syringe. After infiltration, plants were incubated at 22 °C for 44 h and then transferred at the indicated temperatures for 4 h until LUC activity was measured. For examining the interactions in Fig. 2b, the indicated Nluc and Cluc construct combinations were transiently expressed in N. benthamiana at 22 °C for 2 d. For examining the temperature-dependent associations of OsMS1 and OsMS1^wenmin1 with TDR in LCI assays in Fig. 4b, N. benthamiana plants infiltrated with the Agrobacteria combinations were incubated at 22 °C for 44 h and then transferred to 30 °C or not for another 4 h.

To detect luciferase activity, 1 mM D-luciferin solution (Promega, Cat. No: E1602) was sprayed onto the leaves of N. benthamiana, followed by incubation in darkness for 5–10 min. A low-light cooled CCD imaging apparatus (NightOWL II LB983 with Indigo software) was used to capture the image. An exposure time of 5 min was used to take images.

To quantify protein–protein interaction intensity, leaf discs infiltrated with the desired Agrobacterium combinations were taken and incubated with 50 μL water containing 1 mM D-luciferin in a Costar^® 96-Well White Flat Bottom Polystyrene High Bind Microplate (Corning, Cat. No: 7400) for 5–10 min. The luminescence activity was measured using the GLOMAX 96 microplate luminometer (Promega, USA). The protein–protein interaction intensity was shown as relative luminescence unit (RLU). To avoid the bias of transfection efficiency, we used at least twelve individual leaves of six tobacco plants for each combination.

Luciferase assay

For detecting the temperature-dependent protein abundance, 35S:OsMS1 or 35S:OsMS1^wenmin1 were cloned into vector pGreenII 0800-LUC⁶² using primers pGreenII 0800-OsMS1-LUC-F and pGreenII 0800-OsMS1-LUC-R. 35S:TDR were cloned into vector pGreenII 0800-LUC⁶² using primers pGreenII 0800-TDR-LUC-F and pGreenII 0800-TDR-LUC-R, and used as a control. Primers are summarized in Supplementary Data 1. All the constructs were introduced into A. tumefaciens strain GV3101.

The Agrobacterium bacteria were activated and then infiltrated into N. benthamiana plants as LCI assay. After infiltration, plants were incubated at 22 °C for 44 h and then transferred to the indicated temperatures. The firefly and Renilla luciferase activity were detected using the Dual-Luciferase® Reporter Assay System (Promega, Cat. No: E1960) in the GLOMAX 96 microplate luminometer (Promega, USA) following the manufacturer’s instructions. The firefly luciferase activity was normalized to the Renilla luciferase activity to control for transfection efficiency. The relative firefly luciferase activity reflects the protein abundance of OsMS1, OsMS1^wenmin1, or TDR at the indicated temperatures. Three independent replicates were performed for each condition.

Confocal microscopy observation

For the subcellular localization of OsMS1 in rice, root tip cells of 7-day-old 35S:GFP-OsMS1 transgenic rice grown at 22 °C were visualized using a laser scanning confocal microscope (Zeiss LSM710 META, Germany). Nuclei were stained blue by 4′,6-diamidino-2-phenylindole (DAPI).

For the subcellular localization of OsMS1 and OsMS1^wenmin1 in Arabidopsis, root tip cells and cotyledons of 4-day-old 35S:GFP-OsMS1 and 35S:GFP-OsMS1^wenmin1 transgenic Arabidopsis plants were visualized using a laser scanning confocal microscope (Nikon confocal microscope A1R, Japan).

For the subcellular localization of OsMS1 and OsMS1^wenmin1 in N. benthamiana, 35S:GFP-OsMS1 + 35S:mCherry or 35S:GFP-OsMS1^wenmin1 + 35S:mCherry were transiently co-expressed in fully expanded leaves of 6-week-old N. benthamiana as described in LCI assay. The GFP and RFP fluorescence signals were visualized using a laser scanning confocal microscope (Zeiss LSM710 META, Germany) after 48–72 h incubation at 22 °C in the dark.

For the subcellular localization of OsMS1 and OsMS1^wenmin1 in rice protoplast, we used the japonica variety ZH11 to prepare and transform rice protoplasts. The plasmids (10 μg per construct) were introduced by PEG-mediated transfection⁶³. The GFP and DAPI signals were visualized using a laser scanning confocal microscope (Zeiss LSM710 META, Germany) after 18 h incubation at 28 °C in the dark.

To examine whether GFP-OsMS1 and GFP-OsMS1^wenmin1 protein levels are temperature-dependent in Fig. 3c, N. benthamiana expressing 35S:GFP-OsMS1 or 35S:GFP-OsMS1^wenmin1 were grown at 22 °C for 44 h then transferred to 30 °C or not for another 4 h. The GFP signal in leaves was visualized under a laser scanning confocal microscope (Zeiss LSM710 META, Germany). The excitation wavelength used for DAPI, GFP/YFP, and mCherry is 405 nm, 488 nm, and 543 nm, respectively. For quantitative analysis of Fig. 3c in Fig. 3d, the GFP-OsMS1 and GFP-OsMS1^wenmin1 protein levels were quantified by comparing the GFP fluorescence intensity of the cell expressing 35S:GFP:OsMS1 or 35S:GFP:OsMS1^wenmin1 with that of RFP fluorescence intensity of 35S:mCherry in the same cell. Fluorescence intensity was determined using ImageJ software. All the images in a single experiment were captured under the same setting parameters. Zen Black 2009 (Zeiss) was used to export pictures taken by Zeiss LSM710 META.

Bimolecular fluorescence complementation assays

To generate the plasmid 35S:OsMS1-cYFP and 35S:OsMS1^wenmin1-cYFP the ORF of OsMS1 and OsMS1^wenmin1 was amplified with the primers OsMS1-YC-F and OsMS1-YC-R and cloned into the pSPYCE(M) vector⁶⁴. To generate the plasmid 35S:H4-nYFP, the ORF of H4 was amplified with the primers H4-YN-F and H4-YN-R and cloned into the pSPYNE173 vector⁶⁴. To generate the plasmid 35S:TDR-nYFP, the ORF of TDR was amplified with the primers TDR-YN-F and TDR-YN-R and cloned into the pSPYNE173 vector⁶⁴. Primers are summarized in Supplementary Data 1. Co-transfection of the indicated constructs (for example, those encoding OsMS1-cYFP and TDR-nYFP) into N. benthamiana leaf epidermal cells was performed as LCI assay. YFP fluorescence was visualized and photographed using a confocal scanning microscope (Zeiss LSM710 META, Germany). The excitation wavelength used for YFP and mCherry is 488 nm and 543 nm, respectively. Each BiFC assay was repeated at least three times. For detecting the interactions of OsMS1 with H4 in Fig. 2c, the indicated nYFP and cYFP construct combinations were transiently expressed in N. benthamiana at 22 °C for 48 h. For detecting the temperature-dependent interactions of OsMS1 and OsMS1^wenmin1 with TDR in Fig. 4d, the co-infiltrated tobacco plants were incubated at 22 °C for 44 h and then transferred to 30 °C for another 4 h or not.

In vitro pull-down assay

To produce GST-tagged OsMS1 and OsMS1^wenmin1 proteins, the full-length coding sequences of OsMS1 and OsMS1^wenmin1 were amplified and cloned into the GST vector pGEX-4T-1. To produce FLAG-tagged TDR protein (referred to as FLAG-TDR), the coding sequence of TDR was amplified and cloned into the vector pET-nT. Primers are summarized in Supplementary Data 1.

To examine protein–protein interactions in Fig. 4e, recombinant proteins were expressed in E. coli BL21 (DE3). Equal amounts (~2 μg) of purified GST, GST-OsMS1 or GST-OsMS1^wenmin1 proteins were incubated with ~2 μg of recombinant FLAG-TDR in a volume of 600 μL in buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, pH 8.0, 1% Triton X-100, 10% glycerol, 1×Complete Protease Inhibitor cocktail, and 1 mM phenylmethylsulfonyl fluoride). 30 μL Glutathione Sepharose 4B (GE Healthcare, USA) was then added to each combined solution with gentle rotation at 4 °C for 30 min. The beads were collected by centrifugation at 23 × g for 1 min and washed at least four times with the wash buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, pH 8.0, 1% Triton X-100, 10% glycerol, 1×Complete Protease Inhibitor cocktail, and 1 mM phenylmethylsulfonyl fluoride). Finally, the bound proteins were eluted by heating the beads in a 1×SDS loading buffer at 95 °C for 5 min. The eluted proteins were separated by a 10% SDS-PAGE and detected by immunoblotting using an anti-GST antibody (Abmart, Cat. No: M20007, dilution, 1:5000), and an anti-FLAG antibody (Abmart, Cat. No: M20008, dilution, 1:5000), respectively. Signals were detected using eECL Western Blot Kit (Cwbiotech, Cat. No: CW0049), and images were scanned using Tanon-4500 (Shanghai, China).

Rice protoplast preparation

We planted ZH11 in growth chamber for two weeks. Approximately 50–200 leaves at the three-leaf stage were treated with macerozyme and cellulase to prepare protoplast. Briefly, the leaves were cutted into ~0.5 mm fragments, and then incubated in an enzyme solution (0.75% Macerozyme R-10, 1.5% Cellulose RS, 0.6 M mannitol, 10 mM MES at pH 5.7, 10 mM CaCl₂, and 0.1% BSA) for 4–5 h in the dark with gentle rotation. After digestion, the same volume of W5 solution (154 mM NaCl, 5 mM KCl, 125 mM CaCl₂, and 2 mM MES at pH 5.7) was added, and followed by vigorous shaking for 10–30 s. Protoplasts were collected by filtering through 40 μm nylon mesh with three to five washes using W5 solution. The pellets were collected by centrifugation at 150 × g for 5 min. After washing twice with W5 solution, the pellets were then resuspended in MMG solution (0.4 M mannitol, 4 mM MES at pH 5.7, and 15 mM MgCl₂) at a concentration of 2× 10⁶ cells mL⁻¹ for subcellular localization or 2 × 10⁷ cells mL⁻¹ for ChIP-qPCR. Protoplasts were further transiently transformed with the indicated constructs by PEG-mediated transfection according to a published protocol⁶³. Briefly, the indicated DNA plasmids (10 μg) were mixed together with 300 μL protoplasts and the freshly prepared 330 μL polyethylene glycol (PEG) solution (0.4 M mannitol, 40% w/v PEG4000, 0.1 M CaCl₂), and then incubated at room temperature for 20 min. After serial dilutions with W5 solution, the transfected protoplasts were resuspended in W5 solution and cultured overnight at 28 °C in the dark.

Western blot analysis

About 0.5 g stage 9 anthers of pOsMS1:OsMS1-GUS and pOsMS1:OsMS1^wenmin1-GUS transgenic plants were used to extract OsMS1 or OsMS1^wenmin1 protein in Figs. 3i, 3j and Supplementary Fig. 14b. Proteins were prepared as co-immunoprecipitation (Co-IP) assay in Fig. 2e, and detected by anti-GUS antibody (Abmart, Cat. No: P26299, dilution, 1:2000) and anti-ACTIN antibody (Abmart, Cat. No: M20009, dilution, 1:5000).

To examine the subcellular localization of OsMS1 and OsMS1^wenmin1 in Supplementary Fig. 10f, nuclear and cytoplasmic fractions were prepared from stage 9 anthers of pOsMS1:OsMS1-FLAG and pOsMS1:OsMS1^wenmin1-FLAG transgenic rice. About 0.5 g anthers were ground into powder in liquid nitrogen and homogenized in 5 mL Honda buffer (20 mM HEPES-KOH at pH 7.4, 1.25% ficoll, 2.5% Dextran T40, 0.44 M sucrose, 10 mM MgCl₂, 0.5% (v/v) Triton X-100, 5 mM DTT, 1 mM PMSF, Roche protease inhibitor cocktail), and then filtered through 62 μm nylon mesh. Triton X-100 was added to a final concentration of 0.5%, and the mixture was incubated on ice for 15 min. The solution was centrifuged at 1500 × g for 10 min, and the supernatant was used to detect the cytoplasmic protein. The remaining pellets were washed with Honda buffer containing 0.1% (v/v) Triton X-100 and then used to extract the nucleus protein. Proteins were detected by western blots with antibody against FLAG (Abmart, Cat. No: M20008, dilution, 1:5000), H4 (Active Motif, Cat. No: 61521, dilution, 1:5000) or BIP (Abcam, Cat. No: ab108615, dilution, 1:5000). H4 and BIP were used as a marker for nuclei and cytoplasm, respectively.

GUS histochemical staining

Transgenic rice plants were planted in the open field until their inflorescence length reached ~0.5 cm, an indicator of anther development stage 3⁷, and then transferred to the growth chamber under the indicated day average temperature (DAT). For GUS-staining, panicles were submerged in buffer containing 50 mM NaPO₄ buffer, 0.4 mM each K₃Fe(CN)₆/K₄Fe(CN)₆, 0.1% (v/v) Triton X-100, and 1 mM X-Gluc and stained at 37 °C until blue color became visible. Then the samples were incubated with 75% ethanol until chlorophyll was removed. The GUS-staining panicles were taken photos using a microscope. Notably, pOsMS1:OsMS1-GUS and pOsMS1:OsMS1^wenmin1-GUS panicles were treated under the same setting parameters. For detecting endogenous OsMS1-GUS proteins in Fig. 1d, pOsMS1:OsMS1-GUS transgenic plants were grown at planted in the open field and then transplanted to 22 °C DAT in growth chamber. For detecting endogenous OsMS1-GUS or OsMS1^wenmin1-GUS proteins in Fig. 3h and Supplementary Fig. 14a, pOsMS1:OsMS1-GUS and pOsMS1:OsMS1^wenmin1-GUS transgenic plants were grown in the open field and then transplanted to 22 °C or 30 °C DAT in growth chamber.

Electrophoretic mobility shift assay

To generate the plasmid MBP-OsMS1 and MBP-OsMS1^wenmin1, the ORF of OsMS1 and OsMS1^wenmin1 was amplified with the primers MBP-OsMS1-F and MBP-OsMS1-R and cloned into the pMAL-c2 vector. To generate the plasmid MBP-TDR, the ORF of TDR was amplified with the primers MBP-TDR-F and MBP-TDR-R and cloned into the pMAL-c2 vector. Primers are summarized in Supplementary Data 1. MBP-OsMS1 and MBP-TDR proteins were expressed and purified as described in in vitro pull-down assay. The oligonucleotide sequences containing E-box sequences in the rice EAT1 promoter were found by manual search in the promoter sequence. Double-stranded biotin-labeled DNA probes were generated by annealing sense and antisense oligonucleotides. Annealing reactions were performed at 95 °C for 10 min with 0.5 °C decrease per minute until 25 °C in a PCR thermocycler.

EMSA was conducted using Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Scientific). EMSA reactions were performed in a 20 μL system containing 10 mM Tris-Hcl pH 7.5, 2.5% glycerol, 1 mM DTT, 50 mM KCl, 5 mM MgCl₂, 50 ng/μL Poly (dI·dC), and 0.05% NP-40. The binding assays of the indicated proteins to the EAT1 promoter were performed by incubating 10 fmol Biotin-dsDNA probe with 1 μg MBP-TDR or MBP-OsMS1 proteins (1 μL) at room temperature for 30 min. To test whether TDR binds to the EAT1 promoter differently with OsMS1 or OsMS1^wenmin1, biotin-labeled probes were incubated with a constant amount of MBP-TDR protein with increasing amounts of MBP-OsMS1, MBP-OsMS1^wenmin1 or MBP. Protein-DNA complexes were resolved on native 9% (w/v) polyacrylamide gels at 0.5 × TBE at 100 V for 90 min at 4 °C. Electrophoresed protein-DNA complexes were transferred to a Hybond N⁺ nylon membrane (Amersham, GE Healthcare Life Sciences) at 100 V for 60 min using 0.5 × TBE at 4 °C. The transferred protein-DNA complexes were further cross-linked to the membrane for 10 min with the membrane facing a transilluminator equipped with 312 nm bulbs. Detection of Biotin-labeled DNA by chemiluminescence was performed following the instructions of the manufacturer.

Chromatin immunoprecipitation and quantitative real-time PCR analysis

The proteins expressed in protoplasts were used for the ChIP assay. In brief, we used ZH11 to prepare rice protoplasts as described in rice protoplast preparation section⁶³. The indicated plasmids with final concentration at 5 μg per 100 μL were introduced by PEG-mediated transfection. ChIP experiments were performed according to the published protocol⁶⁵. Briefly, approximately 10 mL protoplasts were cross-linked with 1% (v/v) formaldehyde in ice for 10 min. The crosslink was stopped with the addition of 0.125 M glycine for 5 min. After shearing chromatin into about 500 bp fragments via sonication, anti-MYC antibody (Abcom, Cat. No: ab32, dilution, 1:200) was used to immunoprecipitate genomic DNA fragments. Immunoprecipitated DNA was extracted with phenol/chloroform and precipitated with ethanol. The ChIP experiments were quantified by qPCR in triplicate with appropriate primers (Supplementary Data 1). Empty MYC vector was used as the control. The relative enrichment of promoter DNA was calculated by the % input method with qPCR analysis.

In Fig. 5f, young panicles of pOsMS1:OsMS1-FLAG and pOsMS1:OsMS1^wenmin1-FLAG transgenic plants grown at 22 °C or 30 °C in growth chamber were used. Approximately 2 g young panicles with stage 9 anthers of the transgenic lines were ground into powder in liquid nitrogen and cross-linked using extraction buffer with 1% (v/v) formaldehyde in ice for 10 min. The crosslink was stopped with the addition of 0.125 M glycine for 5 min. The chromatin was isolated and the immunoprecipitation assay was conducted as described above. Anti-FLAG antibody (Abcom, Cat. No: ab 205606, dilution, 1:200) was used to immunoprecipitate genomic DNA fragments. The ChIP experiments were quantified by qPCR in triplicate with appropriate primers (Supplementary Data 1). The relative enrichment of promoter DNA was calculated by the % input method with qPCR analysis.

Open chromatin detection by MNase digestion

Open chromatin was detected using a published protocol⁶⁶. In brief, ~0.5 g young panicles with stage 9 anthers of ZJ1 and ZJ1-wenmin1 were ground into power in liquid nitrogen and suspended in 10 mL of cold nuclei isolation buffer (1 M hexylene glycol, 20 mM PIPES-KOH at pH 7.6, 10 mM MgCl₂, 1 mM EGTA, 15 mM NaCl, 0.5 mM spermidine, 0.15 mM spermine, 0.5% Triton X-100, 10 mM β-mercaptoethanol and 1× Roche protease inhibitor cocktail) with gentle rotation for 15 min. The suspension was filtered through 30 nm CellTrics, and the elute was centrifuged for 10 min at 1500 × g at 4 °C. The pellets were resuspended as crude nuclei extracts with digestion buffer (40 mM Tris-HCl at pH 7.9, 0.3 M Suc, 10 mM MgSO₄, 1 mM CaCl₂, and 1× Roche protease inhibitor cocktail). Equal aliquot of crude nuclei extracts was subject to digestion with increasing MNase amounts at 30 °C for 20 min and DNA was extracted using phenol:chloroform treatment followed by ethanol precipitation and used for qPCR analysis.

Statistics and reproducibility

All data are shown as the mean ± s.e.m. unless indicated otherwise. Statistical analysis was performed using GraphPad Prism 7 software (GraphPad Software, Inc. San Diego, CA, USA). All details on statistics have been indicated in figure legends. For all quantitative measurements, a normal distribution was assumed. For comparisons between two groups, the two-tailed unpaired Student’s t test was applied. No method was undertaken to calculate statistical power and corresponding sample size. Instead, sample size was determined empirically on the basis of experience with similar assays and from sample sizes generally used by other investigators^5,61. Images were processed and analyzed with Adobe Photoshop and ImageJ.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The authors declare that all data supporting the findings of this study are available within the article and its Supplementary Information files. Sequence details for rice OsMS1, TDR, and EAT1 genes used in this study can be found in China Rice Data Center (https://www.ricedata.cn/gene/) with the following accession numbers: LOC_Os09g27620, LOC_Os02g02820, and LOC_Os04g51070, respectively. Tissue-specific expression patterns were obtained from Botany Array Resource (http://bar.utoronto.ca/efprice/cgi-bin/efpWeb.cgi). Expression profiles were derived from RiceXPro (https://ricexpro.dna.affrc.go.jp/). The primers used in this study are provided as Supplementary Data 1. Source data are provided with this paper.

References

Wing, R. A., Purugganan, M. D. & Zhang, Q. F. The rice genome revolution: from an ancient grain to Green Super Rice. Nat. Rev. Genet. 19, 505–517 (2018).
Article CAS PubMed Google Scholar
Huang, X. H. et al. Genomic architecture of heterosis for yield traits in rice. Nature 537, 629–633 (2016).
Article CAS PubMed ADS Google Scholar
Yuan, L. P. Development of hybrid rice to ensure food security. Rice Sci. 21, 1–2 (2014).
Article ADS Google Scholar
Normile, D. Agricultural research. Reinventing Rice Feed World Sci. 321, 330–333 (2008).
CAS Google Scholar
Zhou, H. et al. RNase Z^S1 processes Ub^L40 mRNAs and controls thermosensitive genic male sterility in rice. Nat. Commun. 5, 4884 (2014).
Article CAS PubMed ADS Google Scholar
Chen, L. T. & Liu, Y. G. Male sterility and fertility restoration in crops. Annu. Rev. Plant Biol. 65, 579–606 (2014).
Article CAS PubMed Google Scholar
Yu, J. et al. Two rice receptor-like kinases maintain male fertility under changing temperatures. Proc. Natl Acad. Sci. USA 114, 12327–12332 (2017).
Article CAS PubMed PubMed Central Google Scholar
Zhou, H. et al. Photoperiod- and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA. Cell Res. 22, 649–660 (2012).
Article CAS PubMed PubMed Central Google Scholar
Lohani, N., Singh, M. B. & Bhalla, P. L. High temperature susceptibility of sexual reproduction in crop plants. J. Exp. Bot. 71, 555–568 (2020).
Article CAS PubMed Google Scholar
Zhang, D. B. & Yang, L. Specification of tapetum and microsporocyte cells within the anther. Curr. Opin. Plant Biol. 17, 49–55 (2014).
Article CAS PubMed Google Scholar
McCormick, S. Male gametophyte development. Plant Cell 5, 1265–1275 (1993).
Article PubMed PubMed Central Google Scholar
Hong, L. et al. MIL2(MICROSPORELESS2) regulates early cell differentiation in the rice anther. N. Phytol. 196, 402–413 (2012).
Article CAS Google Scholar
Nonomura, K. et al. The MSP1 gene is necessary to restrict the number of cells entering into male and female sporogenesis and to initiate anther wall formation in rice. Plant Cell 15, 1728–1739 (2003).
Article CAS PubMed PubMed Central Google Scholar
Kaneko, M. et al. Loss-of-function mutations of the rice GAMYB gene impair alpha-amylase expression in aleurone and flower development. Plant Cell 16, 33–44 (2004).
Article CAS PubMed PubMed Central Google Scholar
Liu, Z., Bao, W., Liang, W., Yin, J. & Zhang, D. Identification of gamyb-4 and analysis of the regulatory role of GAMYB in rice anther development. J. Integr. Plant Biol. 52, 670–678 (2010).
Article CAS PubMed Google Scholar
Jung, K. H. et al. Rice Undeveloped Tapetum1 is a major regulator of early tapetum development. Plant Cell 17, 2705–2722 (2005).
Article CAS PubMed PubMed Central Google Scholar
Li, N. et al. The rice tapetum degeneration retardation gene is required for tapetum degradation and anther development. Plant Cell 18, 2999–3014 (2006).
Article CAS PubMed PubMed Central Google Scholar
Niu, N. N. et al. EAT1 promotes tapetal cell death by regulating aspartic proteases during male reproductive development in rice. Nat. Commun. 4, 1445 (2013).
Article PubMed ADS CAS Google Scholar
Ko, S. S. et al. The bHLH142 transcription factor coordinates with TDR1 to modulate the expression of EAT1 and regulate pollen development in rice. Plant Cell 26, 2486–2504 (2014).
Article CAS PubMed PubMed Central Google Scholar
Fu, Z. Z. et al. The rice basic Helix-Loop-Helix transcription factor TDR INTERACTING PROTEIN2 is a central switch in early anther development. Plant Cell 26, 1512–1524 (2014).
Article CAS PubMed PubMed Central Google Scholar
Li, H. et al. PERSISTENT TAPETAL CELL1 encodes a PHD-Finger protein that is required for tapetal cell death and pollen development in rice. Plant Physiol. 156, 615–630 (2011).
Article CAS PubMed PubMed Central Google Scholar
Peng, Q. et al. A fragment substitution in promoter of MS92/PTC1 causes male sterility in rice. Rice Sci. 27, 396–404 (2020).
Article Google Scholar
Yang, Z. F. et al. OsMS1 functions as a transcriptional activator to regulate programmed tapetum development and pollen exine formation in rice. Plant Mol. Biol. 99, 175–191 (2019).
Article CAS PubMed Google Scholar
Wilson, Z. A., Morroll, S. M., Dawson, J., Swarup, R. & Tighe, P. J. The Arabidopsis MALE STERILITY1 (MS1) gene is a transcriptional regulator of male gametogenesis, with homology to the PHD-finger family of transcription factors. Plant J. 28, 27–39 (2001).
Article CAS PubMed Google Scholar
Ito, T. & Shinozaki, K. The MALE STERILITY1 gene of Arabidopsis, encoding a nuclear protein with a PHD-finger motif, is expressed in tapetal cells and is required for pollen maturation. Plant Cell Physiol. 43, 1285–1292 (2002).
Article CAS PubMed Google Scholar
Ito, T. et al. Arabidopsis MALE STERILITY1 encodes a PHD-type transcription factor and regulates pollen and tapetum development. Plant Cell 19, 3549–3562 (2007).
Article CAS PubMed PubMed Central Google Scholar
Yang, C., Vizcay-Barrena, G., Conner, K. & Wilson, Z. A. MALE STERILITY1 is required for tapetal development and pollen wall biosynthesis. Plant Cell 19, 3530–3548 (2007).
Article CAS PubMed PubMed Central Google Scholar
Gomez, J. F. & Wilson, Z. A. A barley PHD finger transcription factor that confers male sterility by affecting tapetal development. Plant Biotechnol. J. 12, 765–777 (2014).
Article CAS Google Scholar
Zhang, D. et al. Construction of a multicontrol sterility system for a maize male-sterile line and hybrid seed production based on the ZmMs7 gene encoding a PHD-finger transcription factor. Plant Biotechnol. J. 14, 1281–1290 (2017).
Google Scholar
Endo, M. et al. High temperatures cause male sterility in rice plants with transcriptional alterations during pollen development. Plant Cell Physiol. 50, 1911–1922 (2009).
Article CAS PubMed Google Scholar
Qi, Y. B. et al. Fine mapping and candidate gene analysis of the novel thermo-sensitive genic male sterility tms9-1 gene in rice. Theor. Appl. Genet 127, 1173–1182 (2014).
Article CAS PubMed Google Scholar
Plevin, M. J., Mills, M. M. & Ikura, M. The LxxLL motif: a multifunctional binding sequence in transcriptional regulation. Trends Biochem. Sci. 30, 66–69 (2005).
Article CAS PubMed Google Scholar
Zeng, L. et al. Mechanism and regulation of acetylated histone binding by the tandem PHD finger of DPF3b. Nature 466, 258–262 (2010).
Article CAS PubMed PubMed Central ADS Google Scholar
Sanchez, R. & Zhou, M. M. The PHD finger: a versatile epigenome reader. Trends Biochem. Sci. 36, 364–372 (2011).
CAS PubMed PubMed Central Google Scholar
Musselman, C. A. & Kutateladze, T. G. PHD fingers: epigenetic effectors and potential drug targets. Mol. Interventions 9, 314–323 (2009).
Article CAS Google Scholar
Xiao, J., Lee, U. S. & Wagner, D. Tug of war: adding and removing histone lysine methylation in Arabidopsis. Curr. Opin. Plant Biol. 34, 41–53 (2016).
Article CAS PubMed Google Scholar
Lehmann, S. G., Lalli, E. & Sassone-Corsi, P. X-linked adrenal hypoplasia congenita is caused by abnormal nuclear localization of the DAX-1 protein. Proc. Natl Acad. Sci. USA 99, 8225–8230 (2002).
Article CAS PubMed PubMed Central ADS Google Scholar
Kawajiri, K. et al. Role of the LXXLL-motif and activation function 2 domain in subcellular localization of Dax-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1). Mol. Endocrinol. 17, 994–1004 (2003).
Article CAS PubMed Google Scholar
Zhang, J. S., Kalkum, M., Yamamura, S., Chait, B. T. & Roeder, R. G. E protein silencing by the leukemogenic AML1-ETO fusion protein. Science 305, 1286–1289 (2004).
Article CAS PubMed ADS Google Scholar
Kumar, S. V. & Wigge, P. A. H2A.Z-containing nucleosomes mediate the thermosensory response in Arabidopsis. Cell 140, 136–147 (2010).
Article CAS PubMed Google Scholar
Kumar, S. V. et al. Transcription factor PIF4 controls the thermosensory activation of flowering. Nature 484, 242–245 (2012).
Article CAS PubMed PubMed Central ADS Google Scholar
Lee, J. H. et al. Regulation of temperature-responsive flowering by MADS-box transcription factor repressors. Science 342, 628–632 (2013).
Article CAS PubMed ADS Google Scholar
Sengupta, P. & Garrity, P. Sensing temperature. Curr. Biol. 23, R304–R307 (2013).
Article CAS PubMed PubMed Central Google Scholar
Wigge, P. A. Ambient temperature signalling in plants. Curr. Opin. Plant Biol. 16, 661–666 (2013).
Article CAS PubMed Google Scholar
Jung, J. H. et al. Phytochromes function as thermosensors in Arabidopsis. Science 354, 886–889 (2016).
Article CAS PubMed ADS Google Scholar
Legris, M. et al. Phytochrome B integrates light and temperature signals in. Arabidopsis. Sci. 354, 897–900 (2016).
CAS Google Scholar
Zhu, J. K. Abiotic stress signaling and responses in plants. Cell 167, 313–324 (2016).
Article CAS PubMed PubMed Central Google Scholar
Heery, D. M., Kalkhoven, E., Hoare, S. & Parker, M. G. A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387, 733–736 (1997).
Article CAS PubMed ADS Google Scholar
Chen, L. Y., Lei, D. Y., Tang, W. B. & Xiao, Y. H. Thoughts and practice on some problems about research and application of two-line hybrid rice. Rice Sci. 18, 79–85 (2011).
Article Google Scholar
Zhou, H. et al. Development of commercial thermo-sensitive genic male sterile rice accelerates hybrid rice breeding using the CRISPR/Cas9-mediated TMS5 editing system. Sci. Rep.-Uk 6, 37395 (2016).
Article CAS ADS Google Scholar
Zhang, D. B., Luo, X. & Zhu, L. Cytological analysis and genetic control of rice anther development. J. Genet Genomics 38, 379–390 (2011).
Article CAS PubMed Google Scholar
Zhou, T. B., Xiao, H. C., li, D. Y. & Duan, J. X. Breeding of indica TGMS line 87N123 (HengnongS-1) in rice. Hunan Agric. Sci. 6, 17–18 (1988).
Ba, A. N. N., Pogoutse, A., Provart, N. & Moses, A. M. NLStradamus: a simple Hidden Markov Model for nuclear localization signal prediction. BMC Bioinforma. 10, 202 (2009).
Article CAS Google Scholar
Letunic, I. & Bork, P. 20 years of the SMART protein domain annotation resource. Nucleic Acids Res. 46, D493–D496 (2018).
Article CAS PubMed Google Scholar
Tamura, K. et al. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739 (2011).
Article CAS PubMed PubMed Central Google Scholar
Winter, D. et al. An “Electronic Fluorescent Pictograph” browser for exploring and analyzing large-scale biological data sets. PLoS ONE 2, e718 (2007).
Article PubMed PubMed Central ADS CAS Google Scholar
Hiei, Y. & Komari, T. Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nat. Protoc. 3, 824–834 (2008).
Article CAS PubMed Google Scholar
Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).
Article CAS PubMed Google Scholar
Wen, H. et al. ZMYND11 links histone H3.3K36me3 to transcription elongation and tumour suppression. Nature 508, 263–268 (2014).
Article CAS PubMed PubMed Central ADS Google Scholar
Chen, H. M. et al. Firefly luciferase complementation imaging assay for protein-protein interactions in plants. Plant Physiol. 146, 368–376 (2008).
Article CAS PubMed PubMed Central ADS Google Scholar
Liu, Z., Li, N., Zhang, Y. & Li, Y. Transcriptional repression of GIF1 by the KIX-PPD-MYC repressor complex controls seed size in Arabidopsis. Nat. Commun. 11, 1846 (2020).
Article PubMed PubMed Central ADS CAS Google Scholar
Hellens, R. P. et al. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods 1, 13 (2005).
Article PubMed PubMed Central Google Scholar
Lin, Q. P. et al. Prime genome editing in rice and wheat. Nat. Biotechnol. 38, 582–585 (2020).
Waadt, R. et al. Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta. Plant J. 56, 505–516 (2008).
Article CAS PubMed Google Scholar
Yamaguchi, N. et al. PROTOCOLS: chromatin immunoprecipitation from Arabidopsis tissues. Arabidopsis Book 12, e0170 (2014).
Article PubMed PubMed Central Google Scholar
Zhang, L. et al. A natural tandem array alleviates epigenetic repression of IPA1 and leads to superior yielding rice. Nat. Commun. 8, 14789 (2017).
Article CAS PubMed PubMed Central ADS Google Scholar

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Acknowledgements

We thank Jian-Min Zhou for technical assistance in LCI assay, Yuanzhu Yang for providing Zhu1S, and Zhihui Xia for preliminary mapping of OsMS1^wenmin1. This work was supported by the grants from the strategic priority research program of the Chinese Academy of Sciences (XDB27010102 to Y.H.LI., XDA24010101 to P.G.D.), the National Ten-Thousand Talents Program of China to Y.H.LI., the National Natural Science Foundation of China (31871219 and 31771340 to Y.H.LI., 30960192, and 31160278, and 31960421 to Y.H.LUO.), Key Research and Development Program of Hainan (ZDYF2021XDNY165 to Y.H.LI.), Major Science and Technology Program Projects of Hainan Province (ZDKJ202001 to Y.H.LUO), the Natural Science Foundation of Shanghai (19ZR1446600 to S.J.C.), the Natural Science Foundation of Hainan Province (30810 to Y.H.LUO.), and Hainan Yazhou Bay Seed Laboratory Project (B21HJ0220 to Y.H.LI).

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These authors contributed equally: Lunying Wu, Xiaohui Jing.

Authors and Affiliations

College of Tropical Crops, Hainan University, Haikou, 570228, China
Lunying Wu, Danni Zou, Shengjian Huang, Tingbo Zhou & Yuehua Luo
State Key Laboratory of Plant Cell and Chromosome Engineering, CAS Centre for Excellence in Molecular Plant Science, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
Lunying Wu, Baolan Zhang, Ran Xu, Penggen Duan & Yunhai Li
The State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, 100871, China
Xiaohui Jing & Chengcai An
Shanghai Agrobiological Gene Center, Shanghai, 201106, China
Shoujun Chen
Sanya Nanfan Research Institute of Hainan University, Haikou, 570228, China
Yuehua Luo
Hainan Yazhou Bay Seed Laboratory, Sanya, 572025, China
Yuehua Luo & Yunhai Li
College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, 100039, China
Yunhai Li
The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, 100101, China
Yunhai Li

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Lunying Wu
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Xiaohui Jing
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Baolan Zhang
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Shoujun Chen
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Ran Xu
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Penggen Duan
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Danni Zou
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Shengjian Huang
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Tingbo Zhou
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Chengcai An
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Yuehua Luo
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Contributions

Y.H.LI. and X.H.J. conceived this project. L.Y.W., X.H.J., and Y.H.LI. designed experiments. L.Y.W. and X.H.J. performed most of the experiments; B.L.Z. performed rice genetic transformation; S.J.C. X.H.J., and L.Y.W. performed allelic test; L.Y.W., R.X., P.G.D., D.N.Z., and S.J.H. grew plants and conducted primary mapping; T.B.Z. identified the HNS-1 plants. X.H.J., L.Y.W., Y.H.LUO., C.C.A., and Y.H.LI. analyzed and discussed the data. X.H.J., L.Y.W., and Y.H.LI. interpreted results, prepared figures, and wrote the manuscript with input from all other authors.

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Correspondence to Chengcai An, Yuehua Luo or Yunhai Li.

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Wu, L., Jing, X., Zhang, B. et al. A natural allele of OsMS1 responds to temperature changes and confers thermosensitive genic male sterility. Nat Commun 13, 2055 (2022). https://doi.org/10.1038/s41467-022-29648-z

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Received: 10 March 2021
Accepted: 26 March 2022
Published: 19 April 2022
DOI: https://doi.org/10.1038/s41467-022-29648-z

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