In the hybrid rice industry, the efficiency in F₁ seed production determines whether combinations can be widely used. In a traditional hybrid rice system, the restorer (R) line is the pollen donor, whereas the male sterile (MS) line is the pollen acceptor. The hybrid seed can be generated only if the floret opening time (FOT) of these two lines coincides. However, the average FOT of MS lines is usually later than R lines, especially in indica-japonica hybrid combinations, which greatly reduce hybrid seed yield. Yixiang 1A (YX1A) is an elite sterile line widely used in China, but its FOT is very late, resulting in low seed production in its different hybrid combinations, which not only increases the cost of hybrid seed production but also limits its further application.

In this study, we screened an early flowering mutant from the ethyl methanesulfonate mutagenized population of Yixiang 1B (YX1B), the corresponding maintainer line of YX1A. The mutant, early-morning flowering1 (emf1), showed a ~2.5 h earlier flowering than its wild-type (WT), YX1B (Figure 1a and Figure S1). Lodicule is an important organ that controls the opening and closing of rice spikelets (Wang et al., 1991). At the maximum flower opening angle, the area of the emf1 lodicule was significantly larger than WT (Figure 1b). Through water absorption experiments, we found that the lodicule of emf1 absorbs more water and expands quickly compared to WT (Figure 1c-d). Transmission electron microscopy revealed that the cell wall of lodicule of emf1 was more loosen than that of WT (Figure 1e). Pectin, cellulose and hemicellulose, the main components of the cell wall, were significantly reduced in emf1 (Figure 1f-i). Presumably, a decrease in lodicule cell wall components resulted in the loosening of the cell wall, which improved water absorption and expansion of lodicules in emf1.

To identify the causal gene conferring emf1 phenotype, we performed fine mapping and narrowed the candidate gene to a 50-kb region containing eight candidate genes (Figure S2A). Using MutMap, we identified a 14-bp deletion with a high SNP index, which caused a frameshift in LOC_Os01g42520 (Figure 1b and Figure S2B). The 2-kb promoter and coding sequence fragment of this gene from WT was transferred into emf1, and the phenotype was restored in the positive transgenic plants (Figure S2C-D). Thus, LOC_Os01g42520 is the causal gene-regulating emf1 phenotype.

The EMF1 gene encodes an unknown protein, which is predicted to contain a signal peptide and a DUF642 domain (Figure S3A). GUS staining and relative expression analysis showed that EMF1 is a constitutively expressed gene, but preferentially expressed in anther, stigma and lodicule near flowering (Figure S3B-C). A study reported that DUF642 showed preferential expression in the plant cell wall (Xie and Wang, 2016), and consistently, the eGFP subcellular assay revealed that EMF1 protein is located in the cell wall (Figure 1k). Comparative transcriptome analysis at the near-flowering stage from emf1 and WT lodicules revealed that differentially expressed genes were enriched in biological processes related to the cell wall and pectin synthesis (Figure S3D-E). Further relative expression analyses confirmed that many cell wall-building genes were significantly down-regulated in emf1 (Figure S3F). Therefore, EMF1 may regulate the FOT by participating in the synthesis of cell wall components.

Pectin is synthesized and esterified in the Golgi apparatus and secreted to the cell wall to be de-esterified by pectin methylesterase (PME). Consistent with Wang et al. (2022), the degree of pectin methyl esterification was higher, but PME activity was significantly decreased in lodicules of emf1 than WT (Figure S4). We additionally demonstrated that when PMEs were knocked out, the FOT of rice was only 1 h and 20 min earlier (Wang et al., 2022), which was significantly lower than that of EMF1 knockout lines (2.5 h earlier). The gap in FOT reveals that EMF1 may also have an additional pathway to regulate the emf1 phenotype.

We found several other proteins interacting with EMF1 in immunoprecipitation (Figure S5), amongst them, Os01g0944700/OsGLN2 is a characterized gene that participates in the development of rice flowers (Akiyama and Pillai, 2001). Yeast two-hybrid assay showed interaction of GLN2 and EMF1 (Figure 1l). To explore whether EMF1 regulates FOT by interacting with GLN2, we developed transgenic knockout (KO) lines targeting OsGLN2. When OsGLN2 was knocked out in cv. Zhonghua 11 (ZH11), the FOT of positive lines was ~1 h earlier than that of ZH11 (Figure 1m and S6A-C). It has been reported that expression of this fusion protein (OsGLN2-GST) in the prokaryotic system can specifically hydrolyze 1–3,1–6-β-glucanase from Palmiform laminaria (Akiyama and Pillai, 2001). To explore whether GLN2 affects FOT by regulating cell wall components as PMEs do, we measured cell wall components in OsGLN2 knockout lines and ZH11. The contents of cellulose of KO lines were significantly lower than ZH11 (Figure S6F-H). Therefore, we speculated that EMF1 regulates the content of pectin and cellulose in the cell wall by binding both PMEs and GLN2, thus affecting the water absorption of lodicule, which ultimately regulates FOT (Figure 1n).

To explore the breeding potential of EMF1, we generated a YX1A-emf1 line by crossing emf1 with YX1A (Figure S7A-D). The YX1A-emf1 showed a 2–2.5 h earlier FOT than the YX1A. To test EMF1 applications in japonica, we knocked out EMF1 in ZH11 and consistently we observed ~2 h earlier flowering (Figure S7E-G). Actually, the favourable allele of EMF1 may have already been used in japonica FOT improvement through artificial selection. Haplotype analysis of EMF1 in diverse rice germplasms (Zhou et al., 2017) showed a C/T transition (an amino acid flip) in the second exon (Figure 1o). This mutation formed a new haplotype (H6) in tropical japonica and showed a significantly earlier FOT than the major haplotype (H4) in japonica (Figure 1p). The long-range linkage disequilibrium (LD) block and slow decay of extended haplotype homozygosity (EHH) around EMF1 in tropical japonica indicate the selection of the H6 haplotype (Figure S8).

In summary, EMF1 interacts with OsGLN2 to regulate the content of cellulose in the cell wall of the lodicule in addition to the previous interaction between EMF1 and PMEs. The loss of EMF1 function resulted in increased water absorption capacity of lodicule and earlier FOT of rice. Our study provides insights into the regulation of rice FOT and could improve the efficiency of hybrid seed production in desirable male sterile lines.

在杂交水稻产业中，F ₁种子生产效率决定了组合能否广泛应用。在传统的杂交水稻系统中，恢复系 (R) 系是花粉供体，而雄性不育系 (MS) 系是花粉受体。只有当这两个品系的小花开放时间（FOT）重合时，才能产生杂交种子。然而，MS系的平均FOT通常晚于R系，特别是在籼-粳杂交组合，大大降低了杂交种子的产量。宜香1A（YX1A）是我国广泛使用的优良不育系，但其FOT较晚，导致其不同杂交组合的制种率低，不仅增加了杂交制种成本，也限制了其进一步推广应用。

在本研究中，我们从 YX1A 的相应维持系易香 1B (YX1B) 的甲磺酸乙酯诱变群体中筛选出一个早开花突变体。突变体，清晨开花1 （emf1），比其野生型（WT） YX1B 早开花约2.5小时（图1a和图S1）。浆片是控制水稻小穗开合的重要器官（Wang et al.,  1991）。在最大花开角处，emf1浆片的面积显着大于 WT（图 1b）。通过吸水实验，我们发现emf1与 WT 相比，吸收更多的水并迅速膨胀（图 1c-d）。透射电子显微镜显示emf1的浆片细胞壁比 WT 的细胞壁更松散（图 1e）。细胞壁的主要成分果胶、纤维素和半纤维素在emf1中显着减少（图 1f-i）。据推测，浆液细胞壁成分的减少导致细胞壁松动，从而改善了emf1中浆液的吸水和膨胀。

为了鉴定赋予emf1表型的因果基因，我们进行了精细定位并将候选基因缩小到包含 8 个候选基因的 50-kb 区域（图 S2A）。使用 MutMap，我们确定了一个具有高 SNP 指数的 14 bp 缺失，这导致LOC_Os01g42520中的移码（图 1b 和图 S2B）。将来自 WT 的该基因的 2-kb 启动子和编码序列片段转移到emf1 中，并在阳性转基因植物中恢复表型（图 S2C-D）。因此，LOC_Os01g42520是调节基因的因果基因emf1表型。

EMF1基因编码一种未知蛋白质，预计该蛋白质含有一个信号肽和一个 DUF642 结构域（图 S3A）。GUS染色和相对表达分析表明EMF1是一个组成型表达基因，但优先在开花附近的花药、柱头和花核中表达（图S3B-C）。一项研究报告称，DUF642 在植物细胞壁中表现出优先表达（Xie 和 Wang，  2016 年），并且一致地，eGFP 亚细胞测定显示 EMF1 蛋白位于细胞壁中（图 1k）。emf1近开花期的比较转录组分析和 WT 浆液显示差异表达的基因在与细胞壁和果胶合成相关的生物过程中富集（图 S3D-E）。进一步的相对表达分析证实，许多细胞壁构建基因在emf1中显着下调（图 S3F）。因此，EMF1可能通过参与细胞壁成分的合成来调节FOT。

果胶在高尔基体中合成和酯化，分泌到细胞壁上，通过果胶甲酯酶 (PME) 去酯化。与王等人一致。( 2022 )，与WT相比， emf1的浆液中果胶甲酯化程度较高，但PME活性显着降低（图S4）。我们还证明，当 PME 被敲除时，水稻的 FOT 仅提前 1 小时和 20 分钟（Wang等人， 2022 年），显着低于EMF1敲除系（提前 2.5 小时）。FOT 中的差距表明EMF1还可能具有调节emf1表型的额外途径。

我们在免疫沉淀中发现了其他几种与 EMF1 相互作用的蛋白质（图 S5），其中Os01g0944700 / OsGLN2是参与水稻花发育的特征基因（Akiyama 和 Pillai，  2001）。酵母双杂交试验显示 GLN2 和 EMF1 的相互作用（图 1l）。为了探索 EMF1 是否通过与 GLN2 相互作用来调节 FOT，我们开发了靶向OsGLN2的转基因敲除 (KO) 系。当OsGLN2在简历中被淘汰。中华 11（ZH11），正线的 FOT 比 ZH11 早约 1 小时（图 1m 和 S6A-C）。据报道，这种融合蛋白（OsGLN2-GST）在原核系统中的表达可以特异性地水解棕榈状海带中的 1-3,1-6-β-葡聚糖酶（ Akiyama 和 Pillai，  2001）。为了探索 GLN2 是否像 PME 一样通过调节细胞壁成分来影响 FOT，我们测量了OsGLN2敲除系和 ZH11 中的细胞壁成分。KO系的纤维素含量显着低于ZH11（图S6F-H）。因此，我们推测EMF1通过结合 PMEs 和 GLN2 调节细胞壁中果胶和纤维素的含量，从而影响浆液的吸水性，最终调节 FOT（图 1n）。

为了探索EMF1的育种潜力，我们通过将emf1与 YX1A 杂交生成了 YX1A- emf1系（图 S7A-D）。YX1A-emf1 的 FOT 比 YX1A 早 2-2.5 小时。为了测试EMF1在粳稻中的应用，我们敲除ZH11中的 EMF1，并且我们始终观察到开花提前约 2 小时（图 S7E-G）。实际上，EMF1的有利等位基因可能已经通过人工选择用于粳稻FOT改良。不同水稻种质中EMF1的单倍型分析（Zhou et al., 2017 ) 在第二个外显子中显示出 C/T 转换（氨基酸翻转）（图 1o）。该突变在热带粳稻中形成了一种新的单倍型（H6），并且比粳稻中的主要单倍型（H4）显示出明显更早的FOT （图1p）。热带粳稻中EMF1周围的长程连锁不平衡（LD）阻断和扩展单倍型纯合性（EHH）的缓慢衰减表明选择了 H6 单倍型（图 S8）。

综上所述，EMF1与 OsGLN2 相互作用，除了之前 EMF1 和 PME 之间的相互作用外，还可以调节浆囊细胞壁中纤维素的含量。EMF1功能的丧失导致稻谷吸水能力增加和水稻早期FOT。我们的研究提供了对水稻 FOT 调控的见解，并可以提高理想雄性不育系中杂交种子生产的效率。