Mutation in HER1 enhances stigma exsertion and hybrid seed production in rice

Domain: neuroscience
Published: 2026-02-04
Editorial outcome: major_revision

Abstract
Hybrid rice has achieved a significant leap in rice yield, but the low outcrossing rate of sterile lines limits hybrid seed production due to rice’s self-pollinating habits, constraining the further development and widespread adoption of hybrid rice. Stigma exsertion is a critical trait affecting outcrossing rate. Here we characterize a rice mutant, high exsertion rate 1 (her1), characterized by enlarged stigma and enhanced stigma exsertion. HER1 encodes a SET domain-containing protein that binds to histone H3 at methylated lysine 9 and regulates H3K9me2 levels. Loss of function of HER1 results in reduced H3K9me2 levels in her1, increasing the transcription of Downstream 2 (DS2), which positively controls stigma size. Using the loss-of-function allele of her1, we develop a high-exsertion male-sterile line herA, which increases hybrid seed production by more than 20% in field conditions, substantiating the breeding value of HER1 in hybrid seed production.

Introduction
Rice (Oryza sativa L.) is a highly self-pollinating crop with flowering traits that favor self-pollination over cross-pollination1. However, hybrid rice seeds are produced when the female parent (male-sterile line) receives pollen from the male parent (restorer line) and undergoes fertilization2. Therefore, the outcrossing rate of the sterile line critically limits hybrid seed production. The outcrossing rate of rice is influenced by both environmental and genetic factors. The environmental effects can be optimized through cultivation techniques3. Genetic factors associated with plant morphology, flowering habits, and the structures of floral organs are all closely related to outcrossing. Stigma size and exsertion rate are key traits determining the outcrossing rate4.

Research on the rice OsPINOID (OsPID) gene has provided some insight into rice stigma formation. Initially discovered in Arabidopsis, PINOID (PID) encodes a serine-threonine protein kinase involved in polar auxin transport5,6. In rice, the reduced function of OsPID results in the absence or diminutive size of stigmas, while its overexpression leads to excessive stigma proliferation, forming up to three or even four stigmas in a single floret, accompanied by a reduced number of stamens7,8. Defective Stigma and Panicle (DSP) encodes an AP2/ERF-domain transcription factor that directly binds to the promoter of OsPID and regulates its transcription levels, influencing rice stigma differentiation9.

Numerous quantitative trait loci (QTL) controlling stigma exsertion have been identified, but only a few genes have been cloned10–20. Grain Size 3 (GS3) is a major gene that controls grain length in rice21,22, and is also the first gene cloned to regulate stigma exsertion. The loss-of-function allele of GS3 not only increases grain length but also elongates the style, making the stigma more likely to extend outside the spikelet, ultimately leading to a significant increase in exsertion rate11. Through a genome-wide association study (GWAS) on 533 accessions, two genes, GS3 and Grain Width 5 (GW5), associated with stigma exsertion were identified23. GW5 encodes a calmodulin-binding protein and acts as a negative regulator of grain width24. The strong functional allele of GW5 can reduce spikelet width, making it easier for stigmas to extend outside the glumes. The haplotype combination GW5gs3 can significantly enhance stigma exsertion as well as the appearance quality of rice grains23. Based on the correlation between grain shape and stigma exsertion, three genes regulating grain size, GS3, Grain Width 8 (GW8), and Grain Shape 9 (GS9), were chosen, and their single and multiple mutant lines were constructed25. Loss of function of these three genes leads to narrower or longer grains21,26,27 and single mutants of all three genes showed enhanced stigma exsertion, an effect that was more pronounced in the double and triple mutants. The outcrossing rate and grain length-to-width ratio of the triple mutant lines were significantly improved25. However, the effects of these genes on enhancing hybrid seed production have not been tested under field conditions.

Epigenetics plays a crucial role in regulating plant growth and development, with histone modification being one of the most critical mechanisms in this regulatory system28. Histone modifications primarily include methylation, acetylation, phosphorylation, and ubiquitination, which are closely associated with gene expression, DNA damage repair and recombination29. The establishment of histone methylation primarily depends on SET domain-containing proteins, which catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) to target lysine residues on histones30. These proteins can be classified into several subfamilies based on their structural characteristics and functions, including SET1, SET2, SU(VAR) 3-9, EZH, and SMYD, among others, which specifically regulate histone methylation modifications at different lysine residues31. H3K9 methylation, especially H3K9me2, is a highly prevalent repressive histone mark in plants and plays crucial roles in maintaining genome stability, regulating transposon activity and controlling gene expression32,33. In Arabidopsis thaliana, the SU(VAR) 3-9 homologs (SUVHs) and SU(VAR) 3-9 related proteins (SUVRs) constitute two important subfamilies of SET domain proteins, which play crucial roles in the establishment of histone H3K9 methylation34. SUVHs possess a unique SRA domain, which is associated with their function to specifically recognize DNA methylation and catalyze histone methylation. For example, SUVH4, also known as KRYPTONITE (KYP), binds to methylated DNA CHG/CHH sites to establish H3K9me235. Unlike SUVHs, SUVR5 has been demonstrated to catalyze H3K9me2 independent of DNA methylation, contributing to repressing gene expression36. H3K9 methylation is closely associated with various important traits in plants, including stress responses and flowering time, among others37–40. However, there are currently no research reports on its relationship with stigma development and stigma exsertion.

Here, we report a gene, High Exsertion Rate 1 (HER1), that regulates stigma exsertion by directly controlling the stigma size, which acts differently from those genes that regulate grain shape. HER1 encodes a SET domain-containing protein that is homologous to SUVR5 in Arabidopsis thaliana. HER1 specifically binds to histone H3 at methylated lysine 9 and regulates H3K9me2 levels. Loss of function of HER1 leads to a reduction in H3K9me2 levels, increasing the transcription of the downstream target, Downstream 2 (DS2), and expanding stigma size. Using the loss-of-function allele of HER1, we develop a high-exsertion male-sterile line herA, which can increase hybrid seed production by more than 20% in field conditions, substantiating the breeding value of HER1 in improving hybrid seed production.

Results
Mutant her1 showed enlarged stigma and increased stigma exsertion rate
We utilized ethyl methanesulfonate (EMS) mutagenesis on the indica three-line hybrid rice maintainer II-32B, which shows purple-black stigmas, and obtained a mutant with a high stigma exsertion rate, her1. Compared to the wild-type II-32B, the stigma exsertion rate of her1 significantly increased from 36.32% to 70.63%, an increase of 94.47% (Fig. 1a, c). Microscopic measurements of pistils revealed that the overall size of the pistil in her1 was larger than that of the wild-type (Fig. 1b). Specifically, the stigma length (brush-like part) increased from 0.96 mm to 1.25 mm, the stigma width (brush-like part) increased from 0.35 mm to 0.45 mm, and the style length (non-brush-like part) increased from 0.78 mm to 0.87 mm (Fig. 1d–f). Stigma exsertion is a key factor in determining the outcrossing rate and hybrid seed production. In her1, the stigma exsertion rate was significantly increased by nearly 100% compared to the wild-type, suggesting that the gene controlling this mutant trait could have high breeding value.

To evaluate the impact of gene mutation on the growth and development of her1 plants, we conducted agronomic trait assessments on both wild-type II-32B and her1. Compared to the wild-type, the grain size of her1 was slightly smaller, with grain length reduced by 0.16 mm (1.88%) and grain width reduced by 0.07 mm (2.02%) (Fig. 1g–i). But the ratio of grain length to width remained unchanged (Supplementary Fig. 1a). The seed setting rate of her1 decreased from 78.48% to 71.29% (Supplementary Fig. 1b). However, there were no significant differences in panicle number per plant (tiller number), panicle length, and grain number per panicle between her1 and the wild-type (Supplementary Fig. 1c–e). The reduction in grain size resulted in a 1.35 g (5.33%) decrease in the thousand-grain weight of her1 (Supplementary Fig. 1f). Potassium iodide staining was used to investigate whether the change in the seed setting rate of her1 was related to its pollen fertility, but there was no significant difference between II-32B and her1 (Supplementary Fig. 2).

To further explore the stigma trait variations in her1, we observed the stigmas of both wild-type and her1 using scanning electron microscopy (SEM). The density and thickness of stigma brushes in her1 were increased compared to the wild-type, and the stigma of her1 appeared overall more robust and substantial (Fig. 1j, k). In the style, the epidermal cells of her1 showed notable protrusions, appearing plumper compared to the wild-type (Fig. 1l, m). The length of the epidermal cells in her1 styles decreased while their width increased, making them shorter and wider compared to the wild-type (Fig. 1n, o). These traits indicate that the increased number and thickened size of the stigma brushes in her1 are the primary factors contributing to the increase in stigma volume. Moreover, it can be inferred that the increased total length of the her1 stigma is mainly caused by an increase in the number of longitudinal cells in the style rather than changes in cell morphology.

HER1 encodes a SET domain-containing protein
To determine the inheritance pattern of the mutant traits in her1, we conducted reciprocal crosses between her1 and the wild-type II-32B. The BCF1 plants exhibited the wild-type phenotype regardless of the direction of the cross, and the segregation patterns of two BCF2 populations both followed a 3:1 ratio (Supplementary Table 1), suggesting that the mutant traits of her1 is governed by a single recessive nuclear gene.

We screened 50 extreme mutant-phenotype individuals from the BCF2 population for whole-genome sequencing and MutMap analysis. The SNP-index map showed a significant linkage peak on chromosome 2, containing four SNPs with an SNP-index of 1.0 (Fig. 2a). SNP1 and SNP2 were located in intergenic and promoter regions, respectively. SNP3 and SNP4 fell within coding regions, while SNP4 results in a synonymous mutation, which does not have a functional effect (Supplementary Table 2). SNP3 was located in the coding region of the gene LOC_Os02g47900, named HER1, which was considered the candidate gene controlling the mutant traits. HER1 is comprised of seven exons, and in her1, the 97th base in exon 4 is mutated from A to T, causing a change in the encoded amino acid from arginine (AGA) to a stop codon (TGA) (Fig. 2b). HER1 encodes a SET domain-containing protein, also known as SET Domain Group 706 (SDG706). The mutation in her1 leads to the truncation of the SET domain and complete loss of the post-SET domain (Fig. 2c).

HER1 was constitutively expressed in rice plants, with expression observed in the roots, leaves, panicles, glumes, pistils, and stamens. Its expression level in the pistil was higher than that in other tissues (Supplementary Fig. 3). To validate the biological function of HER1 in regulating stigma size and exsertion, we constructed a CRISPR/Cas9 knockout vector and a functional complementation vector for HER1, which were introduced into the II-32B and her1 backgrounds, respectively (Supplementary Fig. 4a, b). The phenotypes of the two knockout lines were similar to those of her1, exhibiting significantly increased stigma size and exsertion rate (Fig. 2d–g). Conversely, the phenotypes of the two complementation lines in the her1 background were restored to the wild-type level, with no significant differences in either stigma length or exsertion rate (Fig. 2d–g). These results demonstrate that the traits of her1 are caused by the mutation of HER1, which has a biological function in regulating stigma size and exsertion.

We investigated the biological effects of HER1 in the japonica rice variety Zhonghua 11 (ZH11). The two knockout lines her1-zh-ko1/2 in the ZH11 background showed larger stigmas compared to the wild-type (Supplementary Fig. 5a–c), and the stigma exsertion rate significantly increased from 2.48% to 8.66% and 9.75%, respectively, which is more than a threefold increase compared to the wild-type (Supplementary Fig. 5d). In addition, a ubiquitin promoter-driven overexpression vector of HER1 was constructed and introduced into II-32B (Supplementary Fig. 4c, d). Overexpression of HER1 did not alter the stigma size or exsertion rate in transgenic lines (Supplementary Fig. 5e–i). Phenotypic variation only occurs when there is a deficiency of HER1.

HER1 specifically binds to methylated histone H3K9 and regulates H3K9me2 levels
SET domain-containing proteins are universally localized in the nucleus and exhibit histone lysine methyltransferase (HKMT) activity37–40. In the covalent modification of histones, HKMTs typically act as writers, establishing methylation marks on various lysine residues of histones, particularly H3 and H4, thereby regulating chromatin conformation and related processes to influence gene expression41. Using BLASTP, proteins with sequence similarity to HER1 were screened in rice and Arabidopsis. Phylogenetic analysis revealed that HER1 shares sequence similarity with the H3K9 methyltransferase protein families SUVHs and SUVRs in Arabidopsis, and is highly homologous to the SUVR5, suggesting its potential function in the regulation of H3K9 methylation (Supplementary Fig. 6).

To study the molecular function of HER1 protein, we first transiently expressed a 35S promoter-driven HER1-eGFP fusion protein in onion epidermal cells, and localized HER1 to the nucleus, consistent with a previous report42 (Supplementary Fig. 7a). Immunoblotting analyses were performed using specific antibodies targeting different methylation states of various lysine sites on histone H3 in the stigmas of wild-type and her1. The H3K9me2 level in the stigmas of her1 was reduced by more than 1-fold compared to the wild-type, while the levels of H3K9me1, H3K9me3, and other lysine sites showed minor changes (Fig. 3a). Both knockout and overexpression of HER1 affected the H3K9me2 levels in vivo (Fig. 3b). These results indicate that HER1 protein regulates H3K9me2 levels in plants and may influence the expression of downstream genes through this epigenetic mechanism, thereby modulating stigma exsertion rate in rice.

Using recombinant HER1 in methyltransferase assays, neither western blotting nor mass spectrometry (MS) revealed increased H3K9 methylation compared with controls, and no enzymatic activity was detected when HER1 was further tested using a commercial H3K9 methyltransferase activity kit (ab113453, Abcam) (Fig. 3c and Supplementary Fig. 7b–f). All these results are consistent with a previous report42. Therefore, we suggest that HER1 alone may not possess typical HKMT activity but instead interacts with histones through other mechanisms, thereby affecting H3K9me2 levels.

To verify this hypothesis, we performed a GST pull-down assay with GST-HER1 fusion protein, using calf thymus histones or recombinant mononucleosomes as substrates. Calf thymus histones are naturally purified and rich in various covalent modifications, whereas the histones in recombinant mononucleosomes are unmodified. HER1 specifically bound to calf thymus histones but not to recombinant mononucleosomes, suggesting that HER1 may have a preference for specific covalent modifications on histones (Fig. 3d).

We synthesized various biotinylated peptides of histone H3 with different methylation patterns (including H3K4, H3K9, H3K27, H3K36, and unmethylated H3) and performed a peptide pull-down assay using the His-tagged HER1 protein as the substrate. HER1 specifically bound to H3K9me1, H3K9me2, and H3K9me3 peptides but did not bind to H3K4me3, H3K27me3, or H3K36me3 peptides, nor did it bind to unmodified H3 peptides (Fig. 3e). HER1 1-624 protein, expressed using the coding sequence in her1 (Fig. 2c), could not bind to any form of histone H3 peptide (Fig. 3f). To further validate these results, we performed surface plasmon resonance (SPR) analysis using immobilized GST-HER1 protein. Consistent with the peptide pull-down assay, SPR analysis showed that HER1 selectively bound to H3K9me1, H3K9me2, and H3K9me3 peptides, but not to unmodified H3 peptides or peptides methylated at other lysine residues (Fig. 3g, h and Supplementary Fig. 8a–e). The SPR measurements revealed differences in binding affinities among the H3K9-methylated peptides, with H3K9me2 exhibiting the lowest KD value, suggesting a relatively higher binding affinity compared with the other H3K9-methylated peptides (Supplementary Table 3). In addition, we divided HER1 into three parts, HER1-P1, HER1-P2, and HER1-P3 (P3 contains pre-SET, SET, and post-SET domains), and performed peptide pull-down assays with H3, H3K4me3, H3K9me1/2/3, H3K27me3, and H3K36me3 peptides to test the binding region of the HER1 protein (Supplementary Fig. 8f). Among the three fragments, only HER1-P3, which encompasses the entire SET domain architecture, demonstrated binding affinity to H3K9me peptides, but not to peptides with other modification types (Supplementary Fig. 8g). This finding was further validated using His-tagged constructs (Supplementary Fig. 8h). Based on the observed changes in H3K9me2 levels in her1, as well as in knockout and overexpression lines, it can be inferred that although HER1 may lack HKMT activity, it can specifically bind to H3K9me through P3 (containing SET domain) and maintain its methylation status.

HER1 regulates stigma morphology by modulating the transcription of downstream DS2
Based on the effect of HER1 on histone methylation in vivo (Fig. 3a, b), a Cut&Tag assay was performed using the α-H3K9me2 antibody on stigmas of the wild-type and her1. Global H3K9me2 levels exhibited a notable decline in her1 compared to the wild-type (Fig. 4a), aligning with immunoblotting results (Fig. 3a). Differential analysis on the H3K9me2 peaks identified 550 down-regulated peaks and 255 up-regulated peaks in her1 compared to the wild-type, co-locating with 487 and 236 genes, respectively (Fig. 4b, c). A specific polyclonal antibody, α-HER1, was prepared in rabbits for the identification of downstream targets of the HER1 protein. The Cut&Tag analysis with α-HER1 revealed predominant enrichment of HER1 in the promoter, intergenic, and gene body regions (encompassing CDS and introns), with up to 4559 peaks enriched on the promoters, implying an impact of HER1 on gene transcription (Fig. 4d). Moreover, HER1-enriched peaks exhibited a significant overlap with H3K9me2-enriched peaks in the wild-type, indicating a close relationship between HER1 and di-methylation of H3K9 (Fig. 4e).

RNA-seq analyses on stigmas of II-32B and her1 detected 1542 differentially expressed genes (DEGs), with 320 genes down-regulated and 1222 genes up-regulated in her1 compared to the II-32B (Fig. 4f). Considering the repressive effect of H3K9me2 on gene expression34,43–45, we screened genes that were both enriched by HER1 and up-regulated in expression level (in her1), and performed an overlap analysis with genes previously identified in the stigma cell cluster46, identifying five genes as possible downstream targets of HER1 (Fig. 4g and Supplementary Table 4). Principal component analysis (PCA) confirmed the consistency between biological replicates for both Cut&Tag-seq and RNA-seq (Supplementary Fig. 9).

Among these five genes, DS2 (LOC_Os03g02460), which is specifically expressed in stigmas according to the ePlant database47 (https://bar.utoronto.ca/eplant_rice/; Supplementary Fig. 10a), showed significant enrichment of HER1 in both its promoter region and gene body, along with a notable decrease in H3K9me2 levels (Fig. 4h). Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) analysis using the α-HER1 antibody further validated the enrichment of HER1 on DS2 gene, and in the her1 mutant, the enrichment of HER1 on DS2 was significantly reduced due to the loss of key binding site of the HER1 protein (Supplementary Fig. 10b, c). Whole-genome bisulfite sequencing (WGBS) was also performed in both II-32B and her1. Unlike the HER1 binding and H3K9me2 enrichment observed at its gene region (Fig. 4h), DS2 exhibited very low DNA methylation in both II-32B and her1 (Supplementary Fig. 10d). Importantly, no appreciable differences in DNA methylation patterns were observed at the DS2 locus between II-32B and her1, despite clear differences in H3K9me2 levels and DS2 expression. These results suggest that HER1 regulates H3K9me2 and DS2 expression in a DNA methylation-independent manner, akin to what has been reported for SUVR5 in Arabidopsis36.

DS2 encodes a short-chain dehydrogenase/reductase (SDR) with an unknown function. The RT-qPCR assay demonstrated high DS2 expression in the stigma, with a significantly increased expression level in the stigma of her1 compared to that of the wild-type (Fig. 4i). Similar to the her1 mutant, two independent DS2 overexpression lines showed significantly enlarged stigmas, with stigma length increased from 1.41 mm to 1.65 mm and 1.68 mm, respectively (Fig. 5a–c). Stigma exsertion rate of both DS2-OE1 and DS-OE2 also significantly increased compared to the wild-type (Fig. 5d). We further knocked out DS2 in both II-32B and her1 to create ds2 single mutant and her1-ds2 double mutant. The ds2 single mutant showed a significant decrease in both stigma length and stigma width compared to II-32B, while loss of function of DS2 in her1 significantly limited the stigma size (Fig. 5e–g). These results further indicated that HER1 regulates the expression of DS2 by modulating H3K9me2 levels, ultimately controlling the stigma size and exsertion (Fig. 5h).

The her1 allele enhances hybrid seed production
The increased exsertion rate observed in the her1 mutant suggests that HER1 may have significant breeding value. We searched for natural mutations of HER1 in 533 rice accessions23 and found 21 variations in HER1 and its promoter region, including 18 SNPs and 3 InDels, with only six mutations located in the coding region (Supplementary Fig. 11a, b). All variations were categorized into six major haplotypes, named Hap1-Hap6, with 137, 108, 75, 72, 69, and 35 accessions included, respectively (Supplementary Fig. 11a). Only the accessions with Hap2, Hap3, and Hap4 showed an increase in total stigma length compared to those carrying Hap1, while no significant differences were observed for other haplotypes (Supplementary Fig. 11c). Hap2, Hap3, and Hap4 were almost exclusively present in the indica subpopulations (IndI and IndII), whereas Hap1 and Hap6 were predominantly found in the temperate japonica (TeJ) and tropical japonica (TrJ), respectively; Hap5 was more prevalent in TrJ and aus subpopulations, showing minimal presence in indica rice (Supplementary Fig. 11d). To avoid the impacts of genetic background, we compared the phenotypes of accessions carrying Hap2, Hap3, and Hap4 in indica groups, while those with Hap1, Hap5, and Hap6 were analyzed in japonica and aus groups. The results showed no significant differences in total stigma length among Hap2, Hap3, and Hap4, nor were there any differences observed among Hap1, Hap5, and Hap6 (Supplementary Fig. 11e, f).

To further analyze the effects of different HER1 haplotypes, we selected three accessions from each of Hap1-Hap4, which had shown statistically significant phenotypic differences, and performed qPCR analysis of the DS2 gene in their stigmas. Among the analyzed accessions, only one accession, W108 (Hap2), showed decreased expression compared to the three specific accessions, C069 (Hap2), W259 (Hap3), and W272 (Hap3), while no significant differences were observed among other accessions (Supplementary Fig. 11g). This differential expression did not show a clear association with the phenotype. Furthermore, we found that the variation explained by haplotypes was less than the variation explained by subpopulations (Supplementary Table 5). Given this lack of correlation between HER1 haplotypes, DS2 expression, and phenotypes, the increased total stigma length observed in accessions with Hap2, Hap3, and Hap4 may be caused by differences in subpopulation genetic architecture, rather than natural variations in HER1. In addition, nucleotide diversity (π) of HER1 and its flanking regions (50 kb upstream and 50 kb downstream) was analyzed in both cultivated rice (Oryza sativa) and wild rice (Oryza rufipogon). The nucleotide diversity value for HER1 is much lower than its flanking regions in both cultivated and wild populations (Oryza rufipogon), suggesting that the minimal natural variation in the locus of HER1 might be the result of natural selection rather than artificial selection (Supplementary Fig. 11h).

Therefore, we introduced the loss-of-function allele from her1 into the sterile line II-32A (matched with II-32B) through backcross breeding, naming the new line herA. Compared to II-32A, herA exhibited enlarged stigma and a significant increase in stigma exsertion rate, rising from 47.48% to 75.42% (Fig. 6a–c). In 2022, we conducted a preliminary outcrossing experiment on herA and II-32A, using II-32B as the pollen donor. Compared to II-32A, the outcrossing rate of herA increased by 23.03%, and the seed production number per plant increased by 19.16% (Supplementary Fig. 12).

We massively propagated the seeds of herA and II-32A and conducted a field seed production trial in 2023. The experimental field was approximately 65.87 m in length and 6.50 m in width, covering an area of about 428.15 m² (Fig. 6d). To ensure uniform pollen distribution, the trial utilized an interplanting method between the sterile (A) and maintainer (B) lines. Each B row was alternated with two A rows, with each A row containing half a row of herA and half a row of II-32A (Fig. 6e, f). The sterile line herA showed a notable increase in seed production per plant compared to II-32A (Fig. 6g). Statistical analyses revealed that the outcrossing rate of herA increased from 40.80% to 50.37% compared to II-32A (an increase of 23.46%), the seed production yield per field plot (10 plants) increased from 124.57 g to 150.46 g (an increase of 20.79%), and the seed production yield per 666.7 m² increased significantly from 175.76 kg to 215.08 kg, showing a substantial increase of 22.37% (Fig. 6h–j).

As a recessive allele, the negative effects of her1 may be masked by the dominant allele from the restorer line in the hybrid rice (F1) generation48. We generated six hybrid rice combinations by crossing the male sterile lines II-32A and herA with three restorer lines, Shuhui12, Shuhui527, and Fuhui838 (Supplementary Fig. 13a). Agronomic trait analysis revealed that the hybrid rice combinations developed using herA and those using II-32A showed no significant differences (P > 0.05) in plant height, panicle number per plant, panicle length, grain number per panicle, seed setting rate, thousand-grain weight, and yield per plant (Supplementary Fig. 13b, h). Final yield of every field plot was tested, and none of them revealed significant differences between the hybrid rice combinations developed using herA and those using II-32A (Supplementary Fig. 13i). These results confirmed the significant practical value of HER1 and provided precious material resources for high-outcrossing sterile line breeding, contributing to hybrid seed production.

Methods
Plant materials and growth conditions
The rice varieties used in this study mainly include the indica three-line maintainer II-32B and the japonica variety ZH11. II-32B shows purple-black stigmas, facilitating the investigation and screening of stigma-related traits. The her1 mutant was isolated from an EMS-induced mutant library of II-32B. The breeding application utilized several varieties: the sterile line II-32A (corresponding to II-32B), the indica three-line maintainer Shu6B (bred by our team), the indica three-line maintainer 148B (bred by Hunan Taohuayuan Agricultural Technology Co., Ltd.), and the indica two-line sterile Ju182S (bred by Jiangxi Xiannong Seed Industry Co., Ltd.). Shu6B, 148B, and Ju182S all exhibit high combining ability, good quality, and strong resistance. However, their poor outcrossing habits result in low seed production, indicating significant potential for improvement. All of the materials were grown in the experimental bases of Sichuan Agricultural University in Chengdu, Sichuan (summer), and Lingshui, Hainan (winter).

Evaluation of stigma exsertion rate and other traits
The stigma exsertion can be classified into single exsertion and dual exsertion, based on the number of exserted stigmas. The single stigma exsertion rate (SSER) refer to the percentage of spikelets with single exserted stigma relative to the total number of spikelets. Similarly, the dual stigma exsertion rate (DSER) denotes the percentage of spikelets with dual exserted stigmas. The stigma exsertion rate was universally calculated as the total stigma exsertion rate (TSER) in this study, i.e., stigma exsertion rate (%) = TSER (%) = SSER (%) + DSER (%).

The stigmas used for size measurement were obtained from spikelets on the day of flowering. For each line, at least 10 samples were collected and immediately placed under an optical microscope for observation and measurement. Stigma length and width were measured as the length and width of the brush-like part. Style length was measured as the length of the non-brush-like part.

Total stigma length = stigma length + style length (1)

For SEM analysis62, the stigmas were also obtained from spikelets on the day of flowering, but were immediately placed into a 2.5% glutaraldehyde fixative. After 2 h of fixation, the samples were dehydrated in a gradient series of increasing ethanol concentrations (30, 50, 70, 80, 90, 95, and 100%, each for 15 minutes), and then dried with a critical point dryer (K850, Quorum Technologies). Finally, the samples were coated with gold–palladium using a polaron sputter coater (MC1000, Hitachi) and examined using a scanning electron microscope (Gemini SEM 300, Zeiss).

The samples for measurements of grain length, grain width, seed setting rate, panicle number per plant, panicle length, grain number per panicle, and thousand-grain weight were collected from the experimental base in Chengdu. At least 10 biological replicates were randomly selected and measured for each trait.

Genetic analysis and gene mapping
For genetic analysis, her1 and the wild-type II-32B were crossed reciprocally, and the stigma-related phenotypes of BCF1 plants were observed to determine if they were consistent with either the wild-type or the mutant. By self-pollination, BCF2 populations from both reciprocal crosses were obtained. The stigma-related phenotype of each individual in these populations was identified, and the number of plants with wild-type and mutant phenotypes was recorded. Chi-squared tests were used to verify the segregation ratio and determine the inheritance pattern of the mutant phenotype.

For MutMap analysis, leaves of 50 mutant-phenotype plants from the BCF2 population were pooled in equal proportions, forming a mutant bulk for whole-genome sequencing. The wild-type II-32B was also sequenced as a control. High-quality DNA extraction, library construction, and high-throughput sequencing were performed in collaboration with Wuhan IGENEBOOK Biotechnology Co., Ltd. (on the Illumina NovaSeq 6000 sequencing platform). Fastp (v0.23.4)63 was used to remove sequencing adapters and low-quality data. Clean reads were aligned to the reference genome (MSU7.0, rice.uga.edu) using BWA (v0.7.17)64, and potential PCR duplicates were removed using GATK MarkDuplicates (v4.3.0.0). SNP detection, SNP-index calculation, and linkage analysis were performed using the MutMap Pipeline (v2.3.4)65, filtering for loci with a SNP-index of 1 within highly confident intervals. Functional annotation was performed using SnpEff (v5.2)66 to identify candidate genes and their mutation modes in her1. Protein structures and functional domains were analyzed using the InterPro database (www.ebi.ac.uk/interpro).

Vector construction and genetic transformation
To generate the HER1 knockout construct, a multiplex CRISPR/Cas9 expression vector was generated using the toolkit developed by Yaoguang Liu’s group67. Two targets for HER1 and their primers, HER1-KO-Target1-U6a and HER1-KO-Target2-U6b (Supplementary Table 6), were designed using the Target Design68 web tool. Two sgRNA boxes driven by OsU6a and OsU6b promoters were amplified using overlapping PCR, with pYLsgRNA-OsU6a and pYLsgRNA-OsU6b as templates, and inserted into pYLCRISPR/Cas9Pubi-H to obtain the final HER1-KO vector.

To knockout DS2, one target and its primers, DS2-KO-U6a (Supplementary Table 6), were designed. The sgRNA box was amplified using overlapping PCR, with pYLsgRNA-OsU6a as template, and inserted into pYLCRISPR/Cas9Pubi-H to obtain the complete DS2-KO vector.

For the functional complementation assay, the full-length HER1 gene sequence, including 2 kb of its promoter region, was amplified from the genomic DNA of II-32B using the HER1-CP primers with homologous recombination arms (Supplementary Table 6). It was then integrated into pCAMBIA1300 at the EcoRI and SalI sites to construct the HER1-CP vector, using the In-Fusion Snap Assembly Cloning Kit (638946, Takara).

To overexpress HER1, the coding sequence of HER1 was amplified from the cDNA of II-32B stigma using the HER1-OE primers (Supplementary Table 6), and then integrated into pBWA(V)HU-GFP (modified from pCAMBIA1300 with the ubiquitin promoter and a GFP tag, Wuhan BioRun Biosciences Co., Ltd.) to construct the HER1-OE vector.

To overexpress DS2, the coding sequence of DS2 was amplified from the cDNA of II-32B stigma using the DS2-OE primers (Supplementary Table 6), and then integrated into pCAMBIA2300-35s-eGFP (modified from pCAMBIA2300 with the 35s promoter and an eGFP tag) at the BamHI and SalI sites to construct the DS2-OE vector.

The vectors were introduced into rice plants via Agrobacterium tumefaciens-mediated transformation69.

RNA extraction and RT-qPCR
Total RNA was isolated using TRIzol reagent through sample homogenization, chloroform-induced phase separation, and isopropanol precipitation of the aqueous phase, followed by washing the pellet with 75% ethanol70. The first-strand cDNA was synthesized from 1 µg total RNA using the HiScript IV RT SuperMix for qPCR (R423, Vazyme), according to the manufacturer’s instructions. Transcript-specific primers were designed and listed in Supplementary Table 7. RT-qPCR was performed on the QX400 platform (Sichuan Jielaimei Technology Co., Ltd.) using the ChamQ SYBR qPCR Master Mix (Q311, Vazyme), following the manufacturer’s instructions. OsActin1 was used as the internal control. Each sample underwent at least three technical replicates.

Subcellular localization analysis
The coding sequence of HER1 was amplified from the cDNA of II-32B stigma using the HER1-SL primers (Supplementary Table 6), and then integrated into pCAMBIA2300-35s-eGFP (modified from pCAMBIA2300 with the 35s promoter and an eGFP tag) at the BamHI and SalI sites to construct the HER1-SL vector.

The empty vector, pCAMBIA2300-35S-eGFP, and the HER1-SL vector were used for subcellular localization analysis of the HER1 protein. The plasmids were coated onto gold microcarriers and delivered into onion epidermal cells through biolistic acceleration. Specifically, the microcarriers were accelerated by compressed helium gas into the target tissue. The bombarded samples were subsequently maintained in a dark, humid chamber for 24 h before being examined under a fluorescence microscope (BX63, Olympus)71.

SDS-PAGE and immunoblotting
SDS-PAGE was performed using high-quality gradient precast gels (M00928, GenScript), according the manufacturer’s instructions. The samples were denatured in SDS loading buffer (MB01015, GenScript) at 95°C for 10 min, then electrophoresed at a constant voltage of 100 V for 1 h, and transferred to PVDF membrane (Immobilon®-P, Millipore) at a constant current of 280 mA for 2 h. Then the membrane was sequentially blocked at 4°C in 5% milk for 1 h, incubated with the primary antibody at 4°C overnight, and incubated with the secondary antibody at room temperature for 1 h, all on a rocking shaker. Imaging was carried out using ECL Chemiluminescence Kits (E422 and E423, Vazyme) on the e-BLOT Touch Imager (Shanghai e-BLOT Life Science Co., Ltd.).

Analysis of histone methylation levels in vivo
Histone methylation analysis was performed following the previously reported protocol72 with some modifications. In brief, fresh stigmas from different lines were flash-frozen with liquid nitrogen, then quickly ground into powder, and mixed in nuclei extraction buffer (250 mM sucrose; 15 mM piperazine-N,N-bis (pH 6.8); 5 mM MgCl2; 60 mM KCl; 15 mM NaCl; 1 mM CaCl2; 0.5% Triton X-100; 1 mM PMSF; protease inhibitor Cocktail (Roche) at 4°C for 1 h with rotation. Following centrifugation at 4°C and 3000 × g, the pellets were resuspended with 0.2 M HCl, vortexed thoroughly, and incubated on ice for 30 min. After centrifugation at 4°C and 14,000 × g, the supernatant was transferred to a new EP tube, and the pH was adjusted to 8.0 using 1 M Tris-HCl. The samples were denatured and analyzed through SDS-PAGE and immunoblotting. Several antibodies against various lysine sites on histone H3 were used, including H3K4me2 (07-030, Millipore), H3K4me3 (ab8580, abcam), H3K9me1 (07-450, Millipore), H3K9me2 (ab1220, abcam), H3K9me3 (07-442, Millipore), H3K36me1 (ab9048, abcam), H3K36me2 (ab9049, abcam), and H3K36me3 (ab9050, abcam), with α-H3 (ab1791, abcam) as a control, all at a dilution ratio of 1:3000. Fiji (v1.54f)73 was used for grayscale calculations.

Protein expression and purification
To prokaryotically express His-HER1 and GST-HER1 fusion proteins, the coding sequence of HER1 was amplified from the cDNA of rice stigma using His-HER1 and GST-HER1 primers with different homologous recombination arms (Supplementary Table 6), and then integrated into pET-28a and pGEX-4T-1, respectively, using the In-Fusion Snap Assembly Cloning Kit (638946, Takara) to construct the His-HER1 and GST-HER1 vectors. Protein induction was performed using Escherichia coli BL21 (DE3), cultured in 1000 ml of 2×YT medium with 0.2 mM IPTG and the corresponding antibiotics (Kan+ for His-HER1, and Amp+ for GST-HER1). The GST-HER1 protein for methyltransferase activity and GST pull-down assays was purified using Glutathione Beads (SA008005, Changzhou Smart-Lifesciences), and the His-HER1 protein for peptide pull-down assay was purified using the Ni-NTA Pre-Packed Gravity Column (C600791, Sangon Biotech), following the manufacturer’s instructions. His-HER1 was further concentrated and buffer-exchanged with a displacement buffer (20 mM Tris-HCl (pH 8.5); 100 mM NaCl) using the Amicon® Ultra Centrifugal Filter (UFC8030, Millipore). His-HER1 1-624, and truncated HER1 Protein, HER1-P1, HER1-P2, and HER1-P3 (GST and His tagged), were also induced and purified for use in the peptide pull-down assay. HER1 1-624 was coded by the sequence from the her1 mutant and amplified using the His-HER1 1-624 Primers are listed in Supplementary Table 6.

Histone methyltransferase activity assay
In vitro histone methyltransferase activity assays were performed following a method74 with modifications. 2 μg of protein, 2 μg of substrate, and 0.1 mM of S-adenosylmethionine (SAM) were incubated in a 50 μl mixture with 50 mM Tris-HCl (pH 8.5), 20 mM KCl, 10 mM MgCl2, 100 μM ZnCl2, 10 mM β-mercaptoethanol, and 250 mM sucrose at 37°C for 6 h. Recombinant mononucleosomes, kindly offered by Prof. Aiwu Dong, were used as the substrate, and recombinant G9a protein (31425, Active Motif) served as a positive control. Reaction was stop by incubating at 95°C for 10 min in the SDS loading buffer. SDS-PAGE and immunoblotting were performed as described above. Methylation was detected by mass spectrometry and western blotting using the α-H3K9me2 antibody (ab1220, abcam). For further validation, HKMT activity was also tested using a commercial H3K9 methyltransferase activity kit (ab113453, Abcam), according to the manufacturer’s instructions.

Pull-down assays
In the GST pull-down assay, 20 μl of glutathione beads coupled with GST-HER1 and 2 μg of substrate were incubated in 500 μl of the GST pull-down buffer comprising 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 50 μM ZnCl2 at 4°C with rotation for 2 h. Calf thymus histones (37244-51-2, Sigma-Aldrich) or recombinant mononucleosomes were employed as the substrates. After washing three times with the GST pull-down buffer, the samples were analyzed by SDS-PAGE, Coomassie Brilliant Blue staining, and immunoblotting using α-H3 antibody (ab1791, abcam).

The peptide pull-down assay was performed following a procedure75 with some slight modifications. 20 μl of Pierce Streptavidin Magnetic beads (88816, Thermo Scientific) and 2 μg of biotinylated H3 peptides were first incubated in 500 μl of the peptide pull-down buffer (50 mM Tris-HCl (pH 7.5); 200 mM NaCl; 50 μM ZnCl2; 0.1% Triton X-100) at 4°C with rotation for 2 h. The beads coupled with peptides were subsequently washed three times with 1 ml of peptide pull-down buffer, and incubated with His-HER1 or His-HER1 1-624 in the peptide pull-down buffer at 4°C with rotation for 2 h. After washing three times with peptide pull-down buffer, the samples were analyzed by SDS-PAGE and immunoblotting using α-His antibody (bsm-33004M, Bioss). Amino acid sequences of all the peptides are listed in Supplementary Table 8.

Surface plasmon resonance assays
Surface plasmon resonance (SPR) analysis was performed using a Biacore 1K instrument (Cytiva). GST-HER1 protein was immobilized onto flow cells in a CM5 sensor chip (BR-1005-30, Cytiva) via an amine-coupling kit (BR100050, Cytiva), according to the manufacturer’s instructions. Solvent correction was performed using a running buffer consisting of 1× PBS-P+ supplemented with 5% (v/v) DMSO. Histone H3 peptides, including H3K4me3, H3K9me1, H3K9me2, H3K9me3, H3K36me1, H3K36me2, and unmethylated H3 peptides, were serially diluted in running buffer and injected over the immobilized protein at a flow rate of 30 μL/min with an association time of 60 s. After each injection, the sensor surface was regenerated using 10 mM glycine-HCl (pH 2.0). SPR sensorgrams were globally fitted to a 1:1 Langmuir binding model using Biacore Insight Evaluation software (Cytiva). Amino acid sequences of all the peptides are listed in Supplementary Table 8.

Cut&Tag, RNA-seq and ChIP-qPCR assays
A polyclonal antibody against HER1 was developed by Wuhan Gene Create Biotechnology Co., Ltd. in rabbits, utilizing a synthesized peptide with the sequence NLGKDDGSQK. Cut&Tag assays were performed following a protocol76 with modifications. Briefly, fresh stigmas were collected from pre-flowering spikelets, and each sample was replicated two times biologically. Nuclei were isolated and incubated with primary antibodies against α-HER1 and α-H3K9me2 (ab1220, abcam). After incubation with secondary antibodies, pA-Tn5 transposase was bound to the antibody-chromatin complex. Tagmentation was activated by the addition of Mg2+, and the resulting DNA fragments were purified and amplified via PCR to generate libraries for high-throughput sequencing. The library construction and high-throughput sequencing were performed in collaboration with Wuhan IGENEBOOK Biotechnology Co., Ltd. (on the Illumina NovaSeq 6000 sequencing platform). Fastp (v0.23.4)63 was utilized to remove the adapters low-quality reads, and the clean reads were mapped to the reference genome (MSU7.0, rice.uga.edu) using BWA (v0.7.17)64. Potential PCR duplicates were removed by GATK MarkDuplicates (v4.3.0.0), and MACS (v2.1.1)77 software was utilized for peak calling. PCA was performed by R (4.2.2) to assess the consistency between biological replicates.

For the RNA-seq analyses, fresh stigmas of both wild-type and her1 were collected with three biological replicates for each sample. High-quality RNA extraction, RNA-seq library construction, and high-throughput sequencing were performed in collaboration with Wuhan IGENEBOOK Biotechnology Co., Ltd. (on the Illumina NovaSeq 6000 sequencing platform). Fastp (v0.23.4)63 was used to remove sequencing adapters and low-quality data, and the clean reads were aligned to the reference genome (MSU7.0, rice.uga.edu) using Hisat2 (v2.2.1)78. FeatureCounts (v2.0.6)79 was used to calculate the number of reads mapped to each gene, analyzing the gene expression levels across the entire genome. Then the differentially expressed genes between the wild-type and her1 were detected using edgeR (v3.40.2)80 in R (4.2.2). PCA was performed R (4.2.2) to assess the consistency between biological replicates.

ChIP-qPCR assays were performed following a protocol72 with modifications. Briefly, fresh stigmas of II-32B and her1 were cross-linked with formaldehyde, and chromatin was fragmented into 200–500 bp segments via sonication. The sheared chromatin was then immunoprecipitated using the same antibody used for Cut&Tag. After reversal of cross-links and DNA purification, the enriched DNA fragments were analyzed by qPCR to determine the relative occupancy of HER1 at target genomic regions. Primers are listed in Supplementary Table 7.

WGBS assays
Fresh stigmas of both wild-type and her1 were collected with two biological replicates for each sample. High-quality DNA extraction, bisulfite treatment, WGBS library construction, and high-throughput sequencing were performed in collaboration with Shanghai BIOZERON Biotechnology Co., Ltd. (on the Illumina NovaSeq 6000 sequencing platform). Fastp (v0.23.4)63 was utilized to remove the adapters low-quality reads, and the clean reads were mapped to the reference genome (MSU7.0, rice.uga.edu) using BSMAP (v2.74). Alignment results were visualized using IGV (v2.16.2).

Haplotype analysis and hybrid seed production trial
The 533 accessions for haplotype analysis were kindly provided by Prof. Qifa Zhang. Genotypes were analyzed and classified into haplotypes using the geneHapR (v1.2.4)81 package in R (4.2.2). Phenotype and subpopulation data were obtained from a previous study23. The genomic data of 28 Oryza rufipogon accessions were obtained from the Rice Super Pan-genome Information Resource Database82 (RiceSuperPIRdb, www.ricesuperpir.com). Nucleotide diversity (π) of 533 rice cultivated rice and 28 Oryza rufipogon accessions was analyzed by PopART (1.7) and visualized using R (4.2.2).

Hybridization was performed using the sterile line II-32A as the female parent and her1 as the male parent. Individuals exhibiting the mutant phenotype in each generation were chosen as male parents and underwent repeated backcrossing to II-32A until the resultant line displayed stable inheritance, complete sterility, and a high stigma exsertion rate. The developed high-exertion sterile line was named herA. A large number of seeds of herA and II-32A were propagated, and a seed production trial was conducted in the experimental base in Chengdu, using II-32B as the male parent.

For consistent pollen distribution, the seed production trial utilized a sterile-maintainer interplanting system with a planting density of 15,000 plants per 666.7 m². In the rows of sterile lines, half of each row is planted with herA and the other half with II-32A. Before reaching the flowering phase, each plant underwent leaf pruning and treatment with 920 gibberellins. At the flowering stage, manual pollination was carried out thrice daily with 30-minute intervals until all lines reached the grain filling stage. Upon full maturity of the sterile lines, an extensive collection of individual herA and II-32A plants was conducted using a multi-point random sampling approach to assess their outcrossing rate and seed production yield.

Three restorer lines, Shuhui12, Shuhui527, and Fuhui838, were used for the development of hybrid rice combinations and yield evaluation. The male sterile lines II-32A and herA were respectively crossed with three restorer lines. The F1 hybrids were planted in field plots with 10 plants × 20 rows (200 plants per field plot), covering an area of approximately 10.66 m2. Plant height, panicle number per plant, panicle length, grain number per panicle, seed setting rate, thousand-grain weight, and yield per plant were measured at least 10 times (biological replicates). In addition, total grain yield was determined for each experimental plot to assess field-level productivity.

Statistics and reproducibility
All statistical analyses were performed using GraphPad Prism (v10.2.3). Numbers (n) of samples or replicates are indicated in the figures or figure legends. For 2-sample comparisons, significance was analyzed using the two-tailed unpaired Student’s t test, with the P-values presented in the figures. For multiple comparisons, significance was analyzed using one/two-way ANOVA. All western blot results are representative of at least three independent experiments with similar results.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript entitled “Mutation in HER1 enhances stigma exsertion and hybrid seed production in rice” identifies a new gene HER1, which encodes a SET domain-containing protein that binds to histone H3 at methylated lysine 9 and regulates H3K9me2 levels. They demonstrated that HER1 could target the downstream gene DS2, a short chain dehydrogenase/reductase, and regulates the transcription level of DS2 by modulating its H3K9me2 levels, ultimately influencing stigma size and exsertion. Notably, they showed that introducing loss-of-function allele of her1 into the sterile line II-32A could significantly increase the outcrossing rate compared to II-32A. This study identified a new promising gene for genetic improvement of rice stigma exsertion to facilitate hybrid rice breeding, however, more molecular and genetic evidence should be provided to consolidate the conclusions fully and justify its publication in Nature Communications.\n\nMajor points\n1. The authors demonstrated that HER1 directly target the downstream gene DS2, and regulates its transcription level, ultimately influencing stigma exsertion. However, the genetic evidence in the current manuscript is insufficient to support this conclusion. To consolidate the genetic relationship between HER1 and DS22, they needed to knock out DS2 or downregulate its expression in her1 background and analyze the phenotype of these lines. In addition, it is better to provide the data of the HER1 overexpression line.\n2. In Fig. 4, the authors performed the CUT&Tag assay but not the ChIP assay, so the figure title needs to be revised. In addition, I strongly recommend a ChIP-qPCR experiment usingα-HER1 antibody to substantiate the direct binding of HER1 to the promoter of DS2.\n3. The authors identified six haplotypes of HER1 in a natural cultivated rice accessions, and showed that accessions with Hap2, Hap3, and Hap4 exhibited longer stigma length than those with Hap1 in Supplementary Fig. 8c, how do these compare to those with Hap5 and Hap6? Additionally, the authors should further examine both the expression and H3K9me2 levels of DS2 among accessions with different haplotypes, this result would provide very strong evidence to confirm the conclusion of this study.\n4. Stigma exsertion is considered to be a key trait in determining domestication of O. rufipogon into the Asian cultivated rice. In general, O. rufipogon, indica and japonica have highest, moderate and lowest level of stigma exsertion, respectively. In this study, the authors found that accessions with Hap2, Hap3, and Hap4 (almost indica varieties) have longer stigma than those with Hap1, Hap5, and Hap6 (predominantly found in japonica and aus varieties). Thus, it will be of great interest to explore the genetic variations of HER1 in wild rice and examine how different haplotype have been artificially selected during rice domestication process.\n\nMinor points\n1. Null mutation of HER1 could not only increase rice stigma exsertion, but also lead to detrimental effects, such as reduced grain size and seed setting rate. It needs concern with regarding the potential impact of utilization of her1 allele on the final yield of hybrid rice.\n2. To make the result clearer in Fig.3a, it is better to label the fold change of histone lysine methylation levels in her1 compared to II-32B in each panel.\n3. The molecular function of DS and its regulatory role in pistil development have not been investigated in this study. Alternatively, the authors could make some reasonable speculations in the Discussion.\n4. The authors claim that introducing her1 allele into II-32A could increase its hybrid seed production by more than 20%. Actually, in this study, this result was obtained from an outcrossing test of herA and the maintainer line, but not the restored line.", "reviewer_2": "Although there have been some reports on the cloning of rice stigma exsertion genes, such as the major effect genes GS3 and GW5 for grain shape, there has been no field experiment to confirm whether these genes can increase hybrid seed yield. In this study, the authors cloned a new gene HER1 that controls rice stigma morphology and exsertion, and found that its loss-of-function allele her1 significantly increased the stigma exsertion rate, outcrossing rate, and hybrid seed yield. In addition, the authors also revealed the epigenetic regulatory mechanism of HER1 affecting rice stigma exsertion and created corresponding genetic materials to confirm it. Undoubtedly, this is an innovative and very meaningful work that will help further increase hybrid rice yield and ensure food security. However, there are still a few minor shortcomings that I hope the authors can improve further.\n1. Line127-129: Why was LOC_Os02g47900 directly identified as the only candidate gene? There is no reasonable explanation.\n2. Line131-134: Regarding the domain structure of the HER1 encoded protein, the authors should explain clearly how the protein domain structure was obtained here in the text or in the Materials and Methods section, such as the website or software used.\n3. In Fig. 4g, Up-regulated DEGs ?", "reviewer_3": "The authors described an EMS screen in which they identified HER1 gene as putatively implicated in regulating stigma size and exsertion. Further analyses of 2 CRISPR-Cas9 derived KO of HER1 gene, as well as its functional complementation confirmed the implication of HER1 protein in the stigma size and exsertion. Importantly, HER1 roles was confirmed both in indica and japonica.\nThe authors then demonstrate that HER1 is a histone methyltransferase that specifically binds and methylate H3 at position K9. The genomic approach then demonstrate that decreased H3K9me2 deposition is observed throughout the genome in the her1 ko, and is accompanied by several hundreds of DEG. A cross-analyses of the genomic data in fine identified 5 putative targets in the stigma, including DS2 gene. They finally confirmed the potential target gene DS2 in stigma size and exsertion, as well as the potential agronomical impact of their discovery on hybrid seed production.\nIt's a well-crafted, easy-to-read story with a potentially interesting impact on agronomy. However, several points need to be better addressed to consider this story sufficiently complete\n1. A description of the known role of SDG706 or their homolog is missing. SDG706 correspond to SUVR5 in Arabidopsis thaliana (see and cite doi: 10.1016/j.bbaexp.2007.04.003) that was already described to alter the expression of a subset of stimulus response genes and modify H3K9me2 (PLoS Genet 2012;8(10):e1002995. doi: 10.1371/journal.pgen.1002995) .These informations should be present in the MS, at least in the discussion.\n2. In A. thaliana, SUVR5 is regulating gene expression and H3K9me2 deposition in a DNA methylation independent manner. Is it also true for SDG706 in rice? To confirm this point, a global DNA methylation analyses should be performed in II-32B, her1 and potentially also in the HER1 over-expressing line. This is an important question because the decoupling of histone modifications and methylation is not so common.\n3. IF DS2 gene is an key target of HER1 HKMT, then\n  its over expression may mimic her1 phenotype,\n  DS2 expression should be lower in overexpression line of HER1\n  a double mutant her1 and ds2 should have the same phenotype than ds2 ko alone\nThe authors should test all 3 hypotheses\n4. 35S promoter is often targeted by silencing mechanisms. Therefore, KO and over expression line of HER1 gene should be validated using the native HER1 antibody used for the Cut&Tag approach.\n5. The exact role of H3K9me2 to understand its function in DS2 expression is missing. Indeed, cut&tag and RNA-seq analyses where performed on fresh stigma, where DS2 is specifically expressed. Although DS2 locus is targeted by HER1, that also potentially deposit H3K9me2 along the promoter and the gene body of DS2, DS2 remains quite highly expressed in the stigma. this observation is counter-intuitive since H3K9me2 is supposed to drastically reduce the expression of the targeted gene. Majority of H3K9me2 methylation is realized by SUVH-type HKMT, mainly on repeated sequences such as Transposable Elements, satellites repeats and inactive ribosomal RNA genes. In that context, the authors should better discuss this aspect and consider the implication of HER1 binding activity, potentially independently on its capacity to deposit H3K9me2. In the Fig2, the authors prepared a HER1-CP1 line and successfully complemented the mutation. By doing a directed mutation of HER-CP1 at the active site of the SET domain, the authors will be able to demonstrate (1) whether the SET activity is required to complement the phenotype, and (2) whether HER1 with a mutated active site still accumulate at DS2 locus in the stigma (even just by ChIP-qPCR).\nThis approach would allow to better characterize the role HER1 as a histone modifier and/or reader.\n6. Better explain the sigles along the MS (-line 48: What means GS3 sigle ?; -line 53: What means GW5 sigle ?; -line 66: :What means HER1 sigle?; -line 70: What mean DS2 sigle?)", "reviewer_4": "Liao et al., provide a important stigma exsertion mutants in rice. Her1 encoded a SET domain protein, which might be involved in histone H3K9me2 and increasing the transcripts of DS2. The mutant phenotype is important for hybrid breeding. The results is potentially interesting in epigenetic field, rice hybrid breeding field, and stigma development field.\nMajor concerns:\n1) The most of concerns of the function of HER1. HER1, SDG706, which is a homology of SUVR5 and It probably not contains very low KMT activity. The western blot might be not sensitive enough. The mass spectrometry and radio activity of SAM might provide the new results. Otherwise, we don’t the real biochemical function of HER1.\n2) There are many homologies of HER1 in rice, the only mutation of SUVR subfamily might be not enough to see any reduction of H3K9me2. In Arabidopsis, SUVH subfamily is much important than SUVR subfamily in H3K9me2. Therefore, the replicates of western blot and negative controls are necessary.\n3)Besides pre-SET, SET, and post SET domain, HER1 harbor C2H2 zinc figure domain. We wonder how HER1 bound to H3K9me1, H3K9me2, and H3K9me3. ITC might help us to answer these questions. Generally, pre-SET, SET, and post SET domain did not show any binding activity to H3K9me1, H3K9me2, and H3K9me3.\n4) Generally, H3K9me2 is located in the heterochromatin, but not in euchromatin. It’s hard to understand analysis of transcription with H3K9me2 and HER1 target. Therefore, the upregulation of DS2 might be second effector, not direct effector by HER1. In another word, the upregulated DS2 might be not caused by the downregulation of H3K9me in her1 plants.\n5) We wonder whether HER1 specifically bind to the DS2 and fulfill the H3K9me2 at this locus. The genetic evidence between HER1 and DS2 is necessary.\n6) The correlation analysis of the biological replicates of Cut&Tag and RNA seq are required in supplemental Figures. Otherwise, we don’t know the real quality.\nMinor concerns:\n1) The biological functions of H3K9 in mammalian and plants are necessary in introduction section.\n2) The functions of DS2 is also needed in introduction.\n3) The structure of HER1 and the phylogenetic tree of HER1 are necessary."}, "author_response": "Response to Reviewers\nReviewer #1 (Remarks to the Author):\nThe manuscript entitled “Mutation in HER1 enhances stigma exsertion and hybrid\nseed production in rice” identifies a new gene HER1, which encodes a SET domain-\ncontaining protein that binds to histone H3 at methylated lysine 9 and regulates\nH3K9me2 levels. They demonstrated that HER1 could target the downstream gene\nDS2, a short chain dehydrogenase/reductase, and regulates the transcription level of\nDS2 by modulating its H3K9me2 levels, ultimately influencing stigma size and\nexsertion. Notably, they showed that introducing loss-of-function allele of her1 into\nthe sterile line II-32A could significantly increase the outcrossing rate compared to II-\n32A. This study identified a new promising gene for genetic improvement of rice\nstigma exsertion to facilitate hybrid rice breeding, however, more molecular and\ngenetic evidence should be provided to consolidate the conclusions fully and justify\nits publication in Nature Communications.\nResponse: Thanks for your insightful comments, which have significantly improved\nour manuscript and enabled us to present more convincing results. We have addressed\nthese specific comments in detail below.\nMajor points\n1. The authors demonstrated that HER1 directly target the downstream gene DS2, and\nregulates its transcription level, ultimately influencing stigma exsertion. However, the\ngenetic evidence in the current manuscript is insufficient to support this conclusion.\nTo consolidate the genetic relationship between HER1 and DS22, they needed to\nknock out DS2 or downregulate its expression in her1 background and analyze the\nphenotype of these lines. In addition, it is better to provide the data of the HER1\noverexpression line.\nResponse: Following your suggestion, we knocked out DS2 in the her1 mutant, while\nalso overexpressing DS2 in the wild-type II-32B. The her1-ds2 double mutant\nexhibited a significant reduction in both stigma length and width, indicating that the\nfunctional loss of DS2 restricts the biological effects of her1 in regulating stigma size\n(Fig. 5e-g in the revised manuscript). Overexpression lines of DS2 showed\nsignificantly increased stigma length and exsertion rate, similar to her1 (Fig. 5a-d in\nthe revised manuscript). These new results strengthen our conclusion that HER1\ntargets DS2 and regulates its transcription level, ultimately influencing stigma\nexsertion.\nAs described in Line 182-186, Page 7 and Supplementary Fig. 5e-i in the revised\nmanuscript, the HER1 overexpression lines showed no significant change in stigma\nmorphology and exsertion rate compared to the wild-type. We hypothesize that rice\nplants might be insensitive to changes in HER1 transcriptional levels, with phenotypic\nvariation occurring only when HER1 is deficient.\n2. In Fig. 4, the authors performed the CUT&Tag assay but not the ChIP assay, so the\nfigure title needs to be revised. In addition, I strongly recommend a ChIP-qPCR\nexperiment using α-HER1 antibody to substantiate the direct binding of HER1 to the\npromoter of DS2.\nResponse: We appreciate your careful review in pointing out this error. We have\ncorrected \"ChIP\" to \"Cut&Tag\" in the title of Fig. 4 in the revised manuscript.\nFollowing your suggestion, we performed ChIP-qPCR assays in both wild-type II-\n32B and her1 using the α-HER1 antibody. HER1 was enriched in multiple regions of\nthe DS2 gene, especially the promoter (site 1), and this enrichment was significantly\ndecreased in the her1 mutant compared to the wild-type (Supplementary Fig. 10b, c\nin the revised manuscript). These results further demonstrate that DS2 is a direct\ndownstream target of HER1.\n3. The authors identified six haplotypes of HER1 in a natural cultivated rice\naccessions, and showed that accessions with Hap2, Hap3, and Hap4 exhibited longer\nstigma length than those with Hap1 in Supplementary Fig. 8c, how do these compare\nto those with Hap5 and Hap6? Additionally, the authors should further examine both\nthe expression and H3K9me2 levels of DS2 among accessions with different\nhaplotypes, this result would provide very strong evidence to confirm the conclusion\nof this study.\nResponse: We apologize for the misunderstanding created for you. In the revised\nmanuscript, we have provided a clearer description of the phenotypic differences\namong Hap1-6: “Only the accessions with Hap2, Hap3, and Hap4 showed an increase\nin total stigma length compared to those carrying Hap1, while no significant\ndifferences were observed for other haplotypes.” (Line 294-296, Page 10 in the\nrevised manuscript)\nFollowing your suggestion, we selected three accessions from each of Hap1-Hap4,\nwhich had shown statistically significant phenotypic differences, and performed\nqPCR analysis of DS2 in their stigmas. Only one accession, W108 (carrying Hap2),\nshowed decreased expression compared to the three specific accessions, C069\n(carrying Hap2), W259 (carrying Hap3), and W272 (carrying Hap3), while no\nsignificant differences were observed among others (Supplementary Fig. 11g in the\nrevised manuscript). This differential expression did not show a clear association\nwith the phenotype. Furthermore, we found that the variation explained by haplotypes\nwas less than the variation explained by subpopulations (Supplementary Table 3 in\nthe revised manuscript). This lack of correlation between HER1 haplotypes, DS2\nexpression, and phenotypes has strengthened our hypothesis that the increased total\nstigma length observed in accessions with Hap2, Hap3, and Hap4 may be caused by\ndifferences in subpopulation genetic architecture, rather than natural variations in\nHER1.\n4. Stigma exsertion is considered to be a key trait in determining domestication of O.\nrufipogon into the Asian cultivated rice. In general, O. rufipogon, indica and japonica\nhave highest, moderate and lowest level of stigma exsertion, respectively. In this study,\nthe authors found that accessions with Hap2, Hap3, and Hap4 (almost indica varieties)\nhave longer stigma than those with Hap1, Hap5, and Hap6 (predominantly found in\njaponica and aus varieties). Thus, it will be of great interest to explore the genetic\nvariations of HER1 in wild rice and examine how different haplotype have been\nartificially selected during rice domestication process.\nResponse: We had analyzed the nucleotide diversity (π) of HER1 and its flanking\nregions (50 kb upstream and 50 kb downstream) in both cultivated rice (Oryza sativa)\nand wild rice (Oryza rufipogon). Compared to the flanking regions, HER1 exhibits\nnotably low nucleotide diversity in both cultivated and wild populations\n(Supplementary Fig. 11h in the revised manuscript). This suggests that the\nminimal natural variation in the locus of HER1 might be the result of natural selection\nrather than artificial selection. This is also the reason we employed the mutant allele\nin her1 rather than natural variations of HER1 for our breeding applications.\nMinor points\n1. Null mutation of HER1 could not only increase rice stigma exsertion, but also lead\nto detrimental effects, such as reduced grain size and seed setting rate. It needs\nconcern with regarding the potential impact of utilization of her1 allele on the final\nyield of hybrid rice.\nResponse: We strongly agree with you that the final yield of hybrid rice must be tested.\nTo address this concern, six hybrid rice combinations were developed by crossing the\nmale sterile lines II-32A and herA with three restorer lines, Shuhui12, Shuhui527, and\nFuhui838 (Supplementary Fig. 13a in the revised manuscript). The hybrid rice\ncombinations developed using herA and those using II-32A showed no significant\ndifferences in plant height, panicle number per plant, panicle length, grain number per\npanicle, seed setting rate, thousand-grain weight, and yield per plant (Supplementary\nFig. 13b-h in the revised manuscript). We also tested the final yield of every field\nplot and found that none of them revealed significant differences between the hybrid\nrice combinations developed using herA and those using II-32A (Supplementary Fig.\n13i in the revised manuscript). These results confirmed that the negative effects of\nher1 may be masked by the dominant allele from the restorer line in the hybrid\ngeneration (F ) and indicated the significant practical value of HER1 in hybrid rice\n1\nproduction.\n2. To make the result clearer in Fig.3a, it is better to label the fold change of histone\nlysine methylation levels in her1 compared to II-32B in each panel.\nResponse: Following your suggestion, we have labeled the fold changes in each panel\nof Fig. 3a in the revised manuscript. This modification provides clearer information\nabout changes in methylation levels observed in our experiment.\n3. The molecular function of DS and its regulatory role in pistil development have not\nbeen investigated in this study. Alternatively, the authors could make some reasonable\nspeculations in the Discussion.\nResponse: Following your suggestion, we have added reasonable speculations on the\nmolecular function of DS2 and its regulatory role in pistil development in the\nDiscussion (Line 392-399, Page 14 in the revised manuscript).\n4. The authors claim that introducing her1 allele into II-32A could increase its hybrid\nseed production by more than 20%. Actually, in this study, this result was obtained\nfrom an outcrossing test of herA and the maintainer line, but not the restored line.\nResponse: Thanks for your insightful comment. The outcrossing between male sterile\nlines and maintainer lines represents an essential reproductive process in hybrid rice\nseed production. We specifically chose the maintainer line as the male parent in our\nexperiments because restorer lines typically have longer growth periods compared to\nmale sterile/maintainer lines, and these differences can fluctuate substantially\ndepending on environmental conditions. By using the maintainer line, we ensured\nsynchronized flowering periods between two parents, which was critical for the\nreliability and reproducibility of our experimental results.\nReviewer #2 (Remarks to the Author):\nAlthough there have been some reports on the cloning of rice stigma exsertion genes,\nsuch as the major effect genes GS3 and GW5 for grain shape, there has been no field\nexperiment to confirm whether these genes can increase hybrid seed yield. In this\nstudy, the authors cloned a new gene HER1 that controls rice stigma morphology and\nexsertion, and found that its loss-of-function allele her1 significantly increased the\nstigma exsertion rate, outcrossing rate, and hybrid seed yield. In addition, the authors\nalso revealed the epigenetic regulatory mechanism of HER1 affecting rice stigma\nexsertion and created corresponding genetic materials to confirm it. Undoubtedly, this\nis an innovative and very meaningful work that will help further increase hybrid rice\nyield and ensure food security. However, there are still a few minor shortcomings that\nI hope the authors can improve further.\nResponse: Thanks for your insightful comments, which have significantly improved\nour manuscript and enabled us to present more convincing results. We have addressed\nthese specific comments in detail below.\n1. Line127-129: Why was LOC_Os02g47900 directly identified as the only candidate\ngene? There is no reasonable explanation.\nResponse: We apologize for our insufficient description. Regarding\nLOC_Os02g47900, we have provided a more comprehensive description for why it\nhas been chosen as the candidate gene (Line 154-156, Page 6 in the revised\nmanuscript).\n2. Line131-134: Regarding the domain structure of the HER1 encoded protein, the\nauthors should explain clearly how the protein domain structure was obtained here in\nthe text or in the Materials and Methods section, such as the website or software used.\nResponse: The details about the website used for protein domain structure analysis\nhave been included in the Methods section (Line 507-508, Page 18 in the revised\nmanuscript).\n3. In Fig. 4g, Up-regulated DEGs ?\nResponse: Thank you for pointing out this error. We did utilize a subset of genes that\nwere up-regulated in the her1 mutant, as the mutation of HER1 enhanced the\nexpression of a series of genes. But there was a clear error in the description in the\nmain text. We have corrected this content and added more detailed descriptions to\navoid any misunderstanding (Line 259-260, Page 9 in the revised manuscript).\nReviewer #3 (Remarks to the Author):\nThe authors described an EMS screen in which they identified HER1 gene as\nputatively implicated in regulating stigma size and exsertion. Further analyses of 2\nCRISPR-Cas9 derived KO of HER1 gene, as well as its functional complementation\nconfirmed the implication of HER1 protein in the stigma size and exsertion.\nImportantly, HER1 roles was confirmed both in indica and japonica.\nThe authors then demonstrate that HER1 is a histone methyltransferase that\nspecifically binds and methylate H3 at position K9. The genomic approach then\ndemonstrate that decreased H3K9me2 deposition is observed throughout the genome\nin the her1 ko, and is accompanied by several hundreds of DEG. A cross-analyses of\nthe genomic data in fine identified 5 putative targets in the stigma, including DS2\ngene. They finally confirmed the potential target gene DS2 in stigma size and\nexsertion, as well as the potential agronomical impact of their discovery on hybrid\nseed production.\nIt's a well-crafted, easy-to-read story with a potentially interesting impact on\nagronomy. However, several points need to be better addressed to consider this story\nsufficiently complete.\nResponse: Thanks for your insightful comments, which have significantly improved\nour manuscript and enabled us to present more convincing results. We have addressed\nthese specific comments in detail below.\n1. A description of the known role of SDG706 or their homolog is missing. SDG706\ncorrespond to SUVR5 in Arabidopsis thaliana (see and cite doi:\n10.1016/j.bbaexp.2007.04.003) that was already described to alter the expression of a\nsubset of stimulus response genes and modify H3K9me2 (PLoS Genet\n2012;8(10):e1002995. doi: 10.1371/journal.pgen.1002995) .These informations\nshould be present in the MS, at least in the discussion.\nResponse: Following your advice, we have added a description of SUVR5 in\nArabidopsis thaliana in both Introduction and Discussion sections (Lines 88-89, Page\n3-4, and Lines 374-377, Page 13 in the revised manuscript), including citations to\nthe suggested literature. Additionally, we gave a brief description of the roles of\nhistone modifications and H3K9 methylation in the Introduction section (Lines 69-92,\nPage 3-4 in the revised manuscript).\n2. In A. thaliana, SUVR5 is regulating gene expression and H3K9me2 deposition in a\nDNA methylation independent manner. Is it also true for SDG706 in rice? To confirm\nthis point, a global DNA methylation analyses should be performed in II-32B, her1\nand potentially also in the HER1 over-expressing line. This is an important question\nbecause the decoupling of histone modifications and methylation is not so common.\nResponse: We appreciate your insightful suggestion. Following this recommendation,\nwe performed Whole Genome Bisulfite Sequencing (WGBS) on stigmas from both\nwild-type (II-32B) and the her1 mutant. In contrast to neighboring genes\n(LOC_Os03g02450 and LOC_Os03g02470), the DS2 gene region exhibits very low\nlevels of DNA methylation, while these neighboring genes show considerable\nenrichment of CG, CHG, and CHH methylation in both wild-type and her1 (Fig. R1).\nImportantly, we did not observe significant differences in DNA methylation patterns\nat the DS2 locus between wild-type and her1, despite the clear differences in\nH3K9me2 levels and DS2 expression (Fig. 4h, i in the revised manuscript). These\nobservations strongly suggest that HER1 regulates H3K9me2 and DS2 expression in a\nDNA methylation-independent manner, similar to what has been reported for SUVR5\nin Arabidopsis.\nFig. R1 | WGBS analysis on stigmas from II-32B and her1. Genome browser tracks\nshowing CG, CHG, and CHH methylation enrichment on DS2 and its neighboring\ngene regions in both II-32B and her1.\n3. IF DS2 gene is an key target of HER1 HKMT, then\n> its over expression may mimic her1 phenotype,\n> DS2 expression should be lower in overexpression line of HER1,\n> a double mutant her1 and ds2 should have the same phenotype than ds2 ko alone\nThe authors should test all 3 hypotheses\nResponse: Following your advice, we knocked out DS2 in the her1 mutant, while also\noverexpressing DS2 in the wild-type II-32B. The her1-ds2 double mutant exhibited a\nsignificant reduction in both stigma length and width, indicating that the\nfunctional loss of DS2 restricts the biological effects of her1 in regulating stigma size\n(Fig. 5e-g in the revised manuscript). Overexpression lines of DS2 showed\nsignificantly increased stigma length and exsertion rate, similar to her1 (Fig. 5a-d in\nthe revised manuscript). These new results strengthen our conclusion that HER1\ntargets DS2 and regulates its transcription level, ultimately influencing stigma\nexsertion.\nThe HER1 overexpression lines showed no significant change in stigma morphology\nand exsertion rate, as well as expression of DS2 compared to the wild-type\n(Supplementary Fig. 5e-i in the revised manuscript, and Fig. R2 below).\nTherefore, we propose that rice plants might be insensitive to changes in HER1\ntranscriptional levels, with phenotypic variation occurring only when HER1 is\nfunctionally compromised.\nFig. R2 | Relative expression of DS2 in the overexpression lines of HER1.\nOsActin1 was used as the internal control. The data are presented as means ± s.d. P\nvalues above the bars were calculated using one-way ANOVA.\n4. 35S promoter is often targeted by silencing mechanisms. Therefore, KO and over\nexpression line of HER1 gene should be validated using the native HER1 antibody\nused for the Cut&Tag approach.\nResponse: We concur with your concerns regarding the 35S promoter, and have\ntherefore employed the ubiquitin promoter to drive HER1 overexpression. The new\nresults are now presented in the revised manuscript, replacing the original 35S-related\ncontent (Line 182-186, Page 7 and Supplementary Fig. 5e-i in the revised\nmanuscript). Following the reviewer's advice, we have validated these\noverexpression lines using Western Blot analysis, confirming the accumulation of\nGFP-HER1 protein in the transgenic plants (Supplementary Fig. 3e in the revised\nmanuscript). Importantly, the ubiquitin-driven HER1 overexpression plants still\nexhibited stigma phenotypes very similar to the wild-type, which further substantiates\nour hypothesis as described previously.\n5. The exact role of H3K9me2 to understand its function in DS2 expression is missing.\nIndeed, cut&tag and RNA-seq analyses where performed on fresh stigma, where DS2\nis specifically expressed. Although DS2 locus is targeted by HER1, that also\npotentially deposit H3K9me2 along the promoter and the gene body of DS2, DS2\nremains quite highly expressed in the stigma. this observation is counter-intuitive\nsince H3K9me2 is supposed to drastically reduce the expression of the targeted gene.\nMajority of H3K9me2 methylation is realized by SUVH-type HKMT, mainly on\nrepeated sequences such as Transposable Elements, satellites repeats and inactive\nribosomal RNA genes. In that context, the authors should better discuss this aspect\nand consider the implication of HER1 binding activity, potentially independently on\nits capacity to deposit H3K9me2. In the Fig2, the authors prepared a HER1-CP1 line\nand successfully complemented the mutation. By doing a directed mutation of HER-\nCP1 at the active site of the SET domain, the authors will be able to demonstrate (1)\nwhether the SET activity is required to complement the phenotype, and (2) whether\nHER1 with a mutated active site still accumulate at DS2 locus in the stigma (even just\nby ChIP-qPCR).\nThis approach would allow to better characterize the role HER1 as a histone modifier\nand/or reader.\nResponse: We appreciate your insightful observation regarding the seemingly counter-\nintuitive relationship between H3K9me2 marks and DS2 expression. Gene expression\nis regulated by a complex interplay of multiple epigenetic and transcriptional factors\nrather than H3K9me2 alone. Additionally, H3K9me2 modulates gene expression\nrather than completely silencing it in all contexts. In other words, H3K9me2 functions\nas a fine-tuning regulator rather than a binary on/off switch. The stigma-specific\nexpression of DS2 likely results from a balance between repressive histone marks\n(such as H3K9me2) and strong tissue-specific transcriptional activators binding to its\npromoter region. In the her1 mutant, the reduction of H3K9me2 on the DS2 promoter\nand gene body may disrupt this finely-tuned balance, allowing for the enhanced\nexpression of DS2 beyond its normal levels.\nThe mutated HER1 protein in the her1 mutant lacks the SET domain, which has been\nconfirmed to be the histone-binding site of HER1 (Fig. 3f and Supplementary Fig. 8\nin the revised manuscript). Therefore, we performed ChIP-qPCR assays in both\nwild-type II-32B and her1 using the α-HER1 antibody. The enrichment of HER1 on\nmultiple sites in the DS2 gene region was significantly decreased in the her1 mutant\ncompared to the wild-type (Supplementary Fig. 10b, c in the revised manuscript).\nThese results demonstrate that HER1 with a mutated active site fails to accumulate at\nthe DS2 locus, further indicating the regulatory effect of HER1 on DS2.\n6. Better explain the sigles along the MS (-line 48: What means GS3 sigle ?; -line 53:\nWhat means GW5 sigle ?; -line 66: :What means HER1 sigle?; -line 70: What mean\nDS2 sigle?)\nResponse: We have revised the manuscript to provide the full names of all gene\nsymbols when they first appear (Line 50-55, Page 2, Line 61 -62, Page 3, and Line\n93-99, Page 4).\nReviewer #4 (Remarks to the Author):\nLiao et al., provide a important stigma exsertion mutants in rice. Her1 encoded a SET\ndomain protein, which might be involved in histone H3K9me2 and increasing the\ntranscripts of DS2. The mutant phenotype is important for hybrid breeding. The\nresults is potentially interesting in epigenetic field, rice hybrid breeding field, and\nstigma development field.\nResponse: Thanks for your insightful comments, which have significantly improved\nour manuscript and enabled us to present more convincing results. We have addressed\nthese specific comments in detail below.\nMajor concerns:\n1) The most of concerns of the function of HER1. HER1, SDG706, which is a\nhomology of SUVR5 and It probably not contains very low KMT activity. The\nwestern blot might be not sensitive enough. The mass spectrometry and radio activity\nof SAM might provide the new results. Otherwise, we don’t the real biochemical\nfunction of HER1.\nResponse: We appreciate your suggestion. The in vitro methyltransferase activity of\nHER1/SDG706 was also tested by a previous study (Ding et al., Plant Sci., 2007,\n10.1016/j.plantsci.2007.03.009) which had been cited in our manuscript (Line 213,\nPage 8). They used radioactive isotope-labeled SAM to conduct in vitro enzyme\nactivity assays and likewise failed to detect methyltransferase activity for\nHER1/SDG706. The consistency between our findings and this independent study\nusing a highly sensitive detection method further validates the reliability of our\nexperimental results.\n2) There are many homologies of HER1 in rice, the only mutation of SUVR\nsubfamily might be not enough to see any reduction of H3K9me2. In Arabidopsis,\nSUVH subfamily is much important than SUVR subfamily in H3K9me2. Therefore,\nthe replicates of western blot and negative controls are necessary.\nResponse: We agree with your concern and have addressed it by performing two\nadditional biological replicates in both wild-type II-32B and the her1 mutant. These\nnew experiments consistently confirmed our previous finding that her1 exhibits a\nnotable decrease in global H3K9me2 levels compared to the wild-type (Fig. R3). This\nreproducible result strengthens our conclusion regarding HER1's role in H3K9\nmethylation regulation.\nFig. R3 | Immunoblotting analysis on H3K9me2 in stigmas of wild-type II-32B\nand the her1 mutant. The numbers represent the fold changes of her1 compared to\nWT, with H3 as control.\n3)Besides pre-SET, SET, and post SET domain, HER1 harbor C2H2 zinc figure\ndomain. We wonder how HER1 bound to H3K9me1, H3K9me2, and H3K9me3. ITC\nmight help us to answer these questions. Generally, pre-SET, SET, and post SET\ndomain did not show any binding activity to H3K9me1, H3K9me2, and H3K9me3.\nResponse: We strongly agree with you that binding site of HER1 to H3K9me must be\ninvestigated. We divided the HER1 protein into three parts, HER1-P1, HER1-P2, and\nHER1-P3 (P3 contains pre-SET, SET, and post-SET domains), and performed peptide\npull-down assays with H3K9me1/2/3 peptides to test the binding region of HER1\n(Supplementary Fig. 8a in the revised manuscript). Among the three fragments,\nonly HER1-P3, which encompasses the entire SET domain architecture, demonstrated\nbinding affinity to H3K9me peptides (Supplementary Fig. 8b in the revised\nmanuscript). These results are consistent with our pull-down experiments using\nHER1-1-624, which is encoded by the coding sequence from her1 mutant that lacks\nthe SET domain, resulting in its inability to bind to H3K9me (Fig. 3f in the revised\nmanuscript). This series of results clearly demonstrates that HER1 interacts with\nH3K9me through its SET domain and regulates H3K9me2 levels.\n4) Generally, H3K9me2 is located in the heterochromatin, but not in euchromatin. It’s\nhard to understand analysis of transcription with H3K9me2 and HER1 target. Therefore,\nthe upregulation of DS2 might be second effector, not direct effector by HER1.\nIn another word, the upregulated DS2 might be not caused by the downregulation of\nH3K9me in her1 plants.\nResponse: Although H3K9 is mainly localized in heterochromatin, studies have\ndemonstrated that H3K9 is involved in regulation of gene expression. For instance,\nSUVR5 functions as an H3K9 methyltransferase, modifying H3K9me2 levels and\ncontrolling a subset of genes involved in environmental stress responses and pathogen\ndefense (Elena et al., PLos Genet., 2012, 10.1371/journal.pgen.1002995).\nAdditionally, our Cut&Tag and ChIP analyses indeed indicate that HER1 is enriched\nat numerous gene loci, and its impact on transcriptional levels is evident, which is also\nconsistent with changes in H3K9me2 levels (Fig. 4, and Supplementary Fig. 10b, c\nin the revised manuscript). Therefore, we still believe that the upregulation of DS2\nwas the direct effect caused by mutation in HER1.\n5) We wonder whether HER1 specifically bind to the DS2 and fulfill the H3K9me2 at\nthis locus. The genetic evidence between HER1 and DS2 is necessary.\nResponse: Following your suggestion, we knocked out DS2 in the her1 mutant, while\nalso overexpressing DS2 in the wild-type II-32B. The her1-ds2 double mutant\nexhibited a significant reduction in both stigma length and width, indicating that the\nfunctional loss of DS2 restricts the biological effects of her1 in regulating stigma size\n(Fig. 5e-g 11 in the revised manuscript). Overexpression lines of DS2 showed\nsignificantly increased stigma length and exsertion rate, similar to her1 (Fig. 5a-d in\nthe revised manuscript). These new results strengthen our conclusion that HER1\ntargets DS2 and regulates its transcription level, ultimately influencing stigma\nexsertion.\n6) The correlation analysis of the biological replicates of Cut&Tag and RNA seq are\nrequired in supplemental Figures. Otherwise, we don’t know the real quality.\nResponse: Following your suggestion, we performed PCA analysis on data from both\nCut&Tag-seq and RNA-seq and confirmed the consistency between biological\nreplicates (Supplementary Fig. 9 in the revised manuscript).\nMinor concerns:\n1) The biological functions of H3K9 in mammalian and plants are necessary in\nintroduction section.\nResponse: Following your advice, we gave a brief description of the roles of\nhistone modifications and H3K9 methylation in the Introduction section (Lines 69-92,\nPage 3-4 in the revised manuscript).\n2) The functions of DS2 is also needed in introduction.\nResponse: We appreciate your suggestion. DS2 encodes an SDR family protein with\nunknown function. SDRs constitute a super family of proteins that are involved in\nvarious biosynthetic and metabolic pathways in plants. Since we have limited\nexperimental evidence on its precise function at this stage, we believe it is more\nappropriate to present our speculations about its function and its potential\ninvolvement in metabolic pathways in the Discussion section rather than the\nIntroduction (Line 392-399, Page 14 in the revised manuscript).\n3) The structure of HER1 and the phylogenetic tree of HER1 are necessary.\nResponse: Following your suggestion, we presented the phylogenetic tree of HER1 in\nthe revised manuscript and clarified the homologous relationship between HER1 and\nArabidopsis SUVR5 (Line 194-199, Page 7, and Supplementary Fig. 6 in the\nrevised manuscript). Additionally, we further elaborated on the reported functions of\nSUVR5, both in the Introduction and Discussion sections (Line 88-91, Page 3-4, and\nLine 374-377, Page 13 in the revised manuscript)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In the revised manuscript, the authors have addressed the feedback provided by the reviewer, and their responses are deemed satisfactory. I have no further comments.", "reviewer_2": "The author answered my questions, please accept.", "reviewer_3": "I am satisfied with the answers given by the authors and agree that this revised version provides evidence that seems to validate the stated hypotheses. I would just remind the authors to also deposit the WGBS data in order to make them publicly accessible.", "reviewer_4": "1) Although the authors put the genetic relationship between HER1 and DS2 in this version. The biological function of HER1 remain unclear, especially for functions of HER1 in histone methylation. Based on western blot and ChIP-seq told the authors that H3K9me2 was reduced in her1, but did not suggest that HER1 is histone methyltransferase for H3K9me2.\nBased on western blot (Figure 3A), we observed that H3K36me1, H3K36me2, H3K9me1, and H3K9me2 were reduced in her1. Therefore, the reduction in H3K9me2 might be the secondary effector by mutations in HER1. The in vitro histone methyltransferase assay using mass spectrometry and radio activity of SAM is necessary. In this version, the author did not provide these important results, and the conclusion based on histone modification need more evidence. Again, Line 208 “These results indicate that HER1 protein regulates H3K9me2 levels in plants ”, the evidence is not enough, based on the reduction of 3K36me1, H3K36me2, H3K9me1, and H3K9me2 in her1.\n2) Figure 3e, The HER1 binding to H3K9me1/2/3 and HER1-P1 binding to H3K9me1/2/3 need more evidence, the only pull- down assay is not reliable. We wonder any special domain in HER1-P1. Again, The binding affinity between H3K9me1/2/3 and HER1-P1 is necessary. The author could not provide how to exclude the no-specific binding. It’s hard to believe these binding based on most histone methyltransferase in plant. The MLL1 binding to H3K4me3 is because of the PHD domain at the N-terminus of MLL1. WDR5 were first reported to bind H3K4me2, this result is easy to understand how MLL1 exhibit H3K4me3 with WDR5. The following study revealed that this binding is based on amino acid at H3, but not on H3K4me2, H3K4me1. Therefore, the binding assay in this manuscript need affinity further examination.\n3) The line 237, “it can specifically bind to H3K9me through the SET domain”, we did not see SET domain has ability to binding to H3K9me in this manuscript.\nOverall, the manuscript is interesting, the detailed experiments are still needed."}, "author_response": "AUTHOR RESPONSES to REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn the revised manuscript, the authors have addressed the feedback provided by the\nreviewer, and their responses are deemed satisfactory. I have no further comments.\nResponse: Thank you very much for your time and positive assessment of our revised\nmanuscript. We are grateful for your constructive comments in the earlier round,\nwhich significantly improved the clarity and rigor of our work. Thank you for your\ncareful review.\nReviewer #2 (Remarks to the Author):\nThe author answered my questions, please accept.\nResponse: Thank you very much for your positive recommendation. Your prior\ncomments helped clarify the text, fix issues, and strengthen the scientific soundness of\nthe work. We appreciate your time and support.\nReviewer #3 (Remarks to the Author):\nI am satisfied with the answers given by the authors and agree that this revised\nversion provides evidence that seems to validate the stated hypotheses. I would just\nremind the authors to also deposit the WGBS data in order to make them publicly\naccessible.\nResponse: Thank you very much for your supportive evaluation. We are glad the\nrevision resolves your comments and more clearly supports the hypotheses. As you\nadvised, we are currently depositing the WGBS data in the SRA database and will\nmake them openly accessible before publication; the accession number will be added\nto the Data Availability section at the proof stage.\nReviewer #4 (Remarks to the Author):\n1) Although the authors put the genetic relationship between HER1 and DS2 in this\nversion. The biological function of HER1 remain unclear, especially for functions of\nHER1 in histone methylation. Based on western blot and ChIP-seq told the authors\nthat H3K9me2 was reduced in her1, but did not suggest that HER1 is histone\nmethyltransferase for H3K9me2.\nBased on western blot (Figure 3A), we observed that H3K36me1, H3K36me2,\nH3K9me1, and H3K9me2 were reduced in her1. Therefore, the reduction in\nH3K9me2 might be the secondary effector by mutations in HER1. The in vitro\nhistone methyltransferase assay using mass spectrometry and radio activity of SAM is\nnecessary. In this version, the author did not provide these important results, and the\nconclusion based on histone modification need more evidence. Again, Line 208\n“These results indicate that HER1 protein regulates H3K9me2 levels in plants”, the\nevidence is not enough, based on the reduction of 3K36me1, H3K36me2, H3K9me1,\nand H3K9me2 in her1.\nResponse: Thank you again for your thoughtful review and insightful suggestions\nregarding the biological function of HER1 and the evidence for its role in histone\nmethylation regulation.\nThe in vitro methyltransferase activity of HER1/SDG706 was previously tested by\nDing et al. (Plant Sci., 2007, 10.1016/j.plantsci.2007.03.009), which has been cited in\nour manuscript (Line 213, Page 8). Using radioactive isotope-labeled SAM, they also\nfailed to detect direct methyltransferase activity for HER1/SDG706 (Fig. R1). The\nconsistency between our findings (Fig. 3c) and this independent study using a highly\nsensitive detection method validates the reliability of our experimental approach.\nThe functional similarity between HER1 and its characterized homolog SUVR5,\nwhich catalyzes H3K9me2 independent of DNA methylation, supports our findings.\nAlthough HER1 does not exhibit direct methyltransferase activity in vitro, it may\nregulate H3K9me2 through alternative mechanisms such as histone\nbinding/maintenance, or serving as a regulatory component of methyltransferase\ncomplexes, as discussed in our manuscript (Discussion Section).\nFurthermore, our Cut&Tag analysis provides compelling evidence for direct\nchromatin regulation. The Venn diagram (Fig. 4e) reveals substantial overlap between\nHER1 binding sites and H3K9me2-marked regions, with 57% of HER1 peaks co-\nlocalizing with H3K9me2 peaks. This high degree of spatial co-localization indicates\ndirect chromatin binding rather than indirect downstream effects.\nCollectively, these findings strongly demonstrate that HER1 plays a direct role in\nH3K9me2 regulation rather than acting solely through secondary mechanisms.\nWe acknowledge that multiple histone modifications are reduced in her1, such as\nH3K36me1, H3K36me2, and H3K9me1. These changes likely represent secondary\nconsequences resulting from the disruption of H3K9me2-mediated chromatin\nstructure rather than direct effects of HER1. While these aspects are intriguing, the\ndetailed investigation of these potential secondary effects is beyond the scope of the\ncurrent study, and we intend to explore these in future studies.\nFig. R1 | In vitro histone methyltransferase activity assay of three rice SET-domain\nproteins, SDG706, SDG714, and SDG704 performed by Ding et al., using radioactive\nisotope-labeled SAM (Ding et al., Plant Sci., 2007, 10.1016/j.plantsci.2007.03.009).\n2) Figure 3e, The HER1 binding to H3K9me1/2/3 and HER1-P1 binding to\nH3K9me1/2/3 need more evidence, the only pull-down assay is not reliable. We\nwonder any special domain in HER1-P1. Again, The binding affinity between\nH3K9me1/2/3 and HER1-P1 is necessary. The author could not provide how to\nexclude the no-specific binding. It’s hard to believe these binding based on most\nhistone methyltransferase in plant. The MLL1 binding to H3K4me3 is because of the\nPHD domain at the N-terminus of MLL1. WDR5 were first reported to bind\nH3K4me2, this result is easy to understand how MLL1 exhibit H3K4me3 with WDR5.\nThe following study revealed that this binding is based on amino acid at H3, but not\non H3K4me2, H3K4me1. Therefore, the binding assay in this manuscript need\naffinity further examination.\nResponse: Thank you for raising these important concerns about the specificity and\nreliability of our peptide-protein binding assays.\nWe initially attempted Isothermal Titration Calorimetry (ITC) as you previously\nsuggested. However, during protein concentration, HER1 consistently formed\naggregates with partial precipitation as the concentration increased, preventing us\nfrom achieving the high concentrations necessary for ITC measurements.\nTo address your concerns, we conducted extensive additional control experiments,\nincluding H3K4me3, H3K27me3, and H3K36me3 peptides beyond H3K9me1/2/3, to\ntest the binding specificity of truncated HER1. Moreover, we used GST-tagged\nconstructs to exclude tag-specific artifacts.\nThe new results consistently show that HER1-P1 fails to bind any tested methylated\npeptides, while HER1-P3 specifically binds to H3K9me1/2/3, but not to other\nmodifications (Supplementary Fig. 8 in the revised manuscript).\nAdditionally, we performed AlphaFold structural prediction analysis of HER1, which\npredicted that the HER1-P3 contains the binding interface for H3K9me2 peptides (Fig.\nR2). This computational prediction is consistent with our experimental observations\nthat only the HER1-P3, but not P1 or P2, exhibits binding activity to H3K9me2.\nThe absence of binding to H3K4me3, H3K27me3, and H3K36me3 peptides provide\nimportant negative controls demonstrating the specificity of HER1-P3-H3K9me\ninteractions. Although quantitative affinity measurements would be ideal, the\nconsistency across multiple experimental approaches with extensive controls provides\nstrong evidence for specific HER1-P3 binding to H3K9 methylated peptides.\nFig. R2 | AlphaFold structural prediction of HER1-H3K9me2 interaction. The P3\nregion (cyan) is predicted to bind H3K9me2 peptide (orange).\n3) The line 237, “it can specifically bind to H3K9me through the SET domain”,\nwe did not see SET domain has ability to binding to H3K9me in this manuscript.\nResponse: Thank you for pointing out this inappropriate statement. We have corrected\nthis sentence to “it can specifically bind to H3K9me through P3 (containing SET\ndomain) …” to accurately reflect our experimental findings (Line 240, Page 9)."}, {"round": 3, "reviewer_comments": {"reviewer_4": "The genetic functions of HER1 is clear. The major concerns are the biochemical functions of HER.\n1)Whether HER1 has histone methylation activity to H3K9 remains unclear in this version. The conclusion of H3K9me methylation of HER1 is based on western blot. We also observed H3K4me3, H3K36me1, and H3K36me2 are reduced in her1 mutant. Therefore, it’s hard to know whether the secondary effectors happened in her1 mutant. If the authors performed ChIP-seq using H3K4me3, H3K36me1, and H3K36me2 antibody, they might find the modifications were reduced at specific sites in her1 mutants. The histone methylation aciviity is necessary.\n2) The results that HER1 protein specifically binds to methylated histone H3K9 is based on western blot. One binding assay is unreliable. The ITC or SPR experiments is necessary."}, "author_response": "AUTHOR RESPONSES to REVIEWER COMMENTS\nReviewer #4 (Remarks to the Author):\nThe genetic functions of HER1 is clear. The major concerns are the biochemical\nfunctions of HER.\nResponse: We greatly appreciate your careful assessment and insightful suggestions\nregarding the biochemical functions of HER1.\n1)Whether HER1 has histone methylation activity to H3K9 remains unclear in this\nversion. The conclusion of H3K9me methylation of HER1 is based on western blot. We\nalso observed H3K4me3, H3K36me1, and H3K36me2 are reduced in her1 mutant. Therefore,\nit’s hard to know whether the secondary effectors happened in her1 mutant. If the\nauthors performed ChIP-seq using H3K4me3, H3K36me1, and H3K36me2\nantibody, they might find the modifications were reduced at specific sites in her1\nmutants. The histone methylation aciviity is necessary.\nResponse: We appreciate your comment regarding the histone methylation activity of\nHER1. The in vitro methyltransferase activity of HER1/SDG706 was previously tested\nby Ding et al. (Plant Sci., 2007, 10.1016/j.plantsci.2007.03.009), which has been cited\nin our manuscript (Line 216, Page 8). Using radioactive isotope-labeled SAM, they\nalso failed to detect direct methyltransferase activity of HER1/SDG706. In response to\nconcerns regarding the sensitivity of our western blot experiments, we further employed\nmass spectrometry (MS) analysis and a histone H3K9 methyltransferase activity\nkit to independently assess the enzymatic activity of HER1. Consistent with our\nwestern blot results, neither MS analysis nor the activity assay kit detected\nmethyltransferase activity of HER1 (Supplementary Fig. 7b-f). Collectively, the\nresults obtained from western blot experiments, MS analysis, and the methyltransferase\nactivity assay kit are fully consistent with the previous report.\nWe also appreciate your concern regarding whether the levels of H3K4me3, H3K36me1,\nand H3K36me2 are reduced. To address this issue, we performed ChIP-seq analyses in\nboth WT and the her1 mutant. Metagene profiling across gene bodies revealed highly\nsimilar distribution patterns and signal intensities for H3K4me3, H3K36me1, and\nH3K36me2 between WT and her1, with no obvious global reduction of these histone\nmarks (Fig. R1). These results indicate that loss of HER1 does not lead to detectable\nchanges in the overall levels or genomic distribution of H3K4me3, H3K36me1, or\nH3K36me2.\nFig. R1 | ChIP-seq metagene profiles of histone methylation marks in II-32B and\nher1. a–d, Metagene analysis showing normalized ChIP-seq read density of total H3\n(a), H3K4me3 (b), H3K36me1 (c), and H3K36me2 (d) across gene bodies in WT (II-\n32B) and her1. Profiles are aligned from 1 kb upstream of the transcription start site\n(TSS) to 1 kb downstream of the transcription termination site (TTS).\n2) The results that HER1 protein specifically binds to methylated histone H3K9 is based\non western blot. One binding assay is unreliable. The ITC or SPR experiments is\nnecessary.\nResponse: We appreciate your comment noting that conclusions drawn from a single\nexperimental approach regarding the specific binding of the HER1 protein to\nmethylated histone H3K9 may lack sufficient rigor. Therefore, we further employed\nsurface plasmon resonance (SPR) assays to examine HER1 binding to different histone\npeptides, including H3K4me3, H3K9me1, H3K9me2, H3K9me3, H3K36me1,\nH3K36me2, and unmethylated H3 peptides. Consistent with our western blot results,\nHER1 specifically bound to methylated H3K9 peptides, including H3K9me1,\nH3K9me2, and H3K9me3, but not to unmodified H3 peptides or to peptides methylated\nat other histone residues (Fig. 3g, h, Supplementary Fig. 8a-e, Supplementary Table\n2, and Fig. R2). These SPR experiments provide independent validation of our findings\nand substantially strengthen our conclusion.\nFig. R2 | Surface plasmon resonance (SPR) analysis of HER1 binding to H3K9me1\n(a), H3K9me2 (b), H3K9me3 (c), H3K4me3 (d), H3K36me1 (e), H3K36me2 (f), and\nunmodified H3 (g) peptides. HER1 was immobilized on the sensor chip, and increasing\nconcentrations (0.3125–10 μM) of peptides were injected. Response units (RU) are\nshown as a function of time."}, {"round": 4, "reviewer_comments": {"reviewer_4": "The authors fixed the most of issues, I don’t have other questions."}, "author_response": "AUTHOR RESPONSES to REVIEWER COMMENTS\nReviewer #4 (Remarks to the Author):\nThe authors fixed the most of issues, I don’t have other questions.\nResponse: We sincerely appreciate your careful assessment and the insightful\nsuggestions that have significantly improved our manuscript."}]

Decision letter