The Xyloglucan Galactosyltransferase <scp>EMT3</scp> Regulates Diurnal Flowering Time by Modulating Lodicule Cell Wall Properties in Rice

Seed production yield is a critical bottleneck limiting the large-scale adoption of hybrid rice technology. Hybrid seed yield mainly depends on the outcrossing traits of the male sterile line, which can be broadly divided into two categories: floret-opening traits and stigma-related traits (Zhou et al. 2017). Among them, diurnal flowering time and stigma exsertion rate are the most decisive factors affecting outcrossing efficiency. However, asynchronous flowering, especially when the male sterile blooms later than the restorer in indica-japonica hybrids, causes poor pollen reception and reduced seed yield. To date, only a few genes regulating diurnal flowering time in rice have been cloned (Gou et al. 2024; Wang et al. 2022; Xu et al. 2022), and the underlying genetic basis and molecular mechanisms remain poorly understood. To identify new regulators of diurnal flowering time, we isolated an early- flowering mutant, early morning flowering time 3 (emt3), from an EMS mutagenized population of Yixiang 1B (YX1B). Compared with its wild-type (WT) YX1B, emt3 initiated flowering about two hours earlier (Figure 1A,B; Figure S1A–C). Lodicules, located between the lemma and palea, are pivotal floral organs that regulate floret opening and closure in rice (Zhang et al. 2025). The lodicules in emt3 were significantly larger than those in WT, both before and during anthesis (Figure 1C; Figure S1A). Transmission electron microscopy further revealed that the lodicule cell walls in emt3 were significantly thinner than those in WT (Figure 1D; Figure S1B). Through water-absorption experiments, we found that the lodicules of emt3 absorbed water more rapidly and expanded faster than those of the WT (Figure S2). Thus, we hypothesize that these structural changes in emt3 facilitate water uptake and expanding, thereby accelerating floret opening early. For genetic mapping of the causal gene for emt3, Bulked segregant analysis (MutMap) identified a G-to-A single-nucleotide variant causing a glutamate-to-lysine substitution (Figure 1E,F; Table S1). This mutation is located in LOC_Os03g05110, which is annotated as a xyloglucan galactosyltransferase (XGT) with high homology to AtMUR3 in Arabidopsis (Figure S4). Mutations in AtGT18, a homologue of AtMUR3 in Arabidopsis, reduce cellulose and hemicellulose content in the cell wall and cause a prostrate growth phenotype (Li et al. 2004). We therefore considered LOC_Os03g05110 as the candidate gene for EMT3. Knockout and overexpression of EMT3 in Zhonghua 11 (ZH11) confirmed its functional role: loss of function accelerated flowering, while overexpression delayed flowering (Figure 1G,H; Figure S5). Thus, LOC_Os03g05110 was validated as EMT3, a regulator of diurnal flowering time in rice. Both qRT-PCR and GUS staining showed that EMT3 is constitutively expressed, with enriched expression in young panicles, stigmas, and pre-flowering lodicules (Figure 1I; Figure S6A). Subcellular localization confirmed that EMT3 resides in the Golgi apparatus (Figure 1J; Figure S6B), consistent with its predicted role in xyloglucan side-chain synthesis (Julian and Zabotina 2022). Enzyme assays showed significantly reduced XGT activity in emt3 lodicules (Figure 1K), accompanied by largely decreased cellulose and hemicellulose content (Figure 1L,M). Using immunofluorescence with the LM15 monoclonal antibody, which specifically recognises the XXXG oligosaccharide epitope of xyloglucan, we observed lower fluorescence in emt3 lodicule cells compared to WT both 1 day before and during flowering (Figure 1N; Figure S7). These findings demonstrate that the emt3 mutation impairs xyloglucan synthesis with reduced cellulose and hemicellulose, leading to thinner and more elastic cell walls that absorb water readily, thereby accelerating lodicule swelling and floret opening. To assess the breeding potential of emt3, we compared the agronomic traits of WT with emt3 plants. The emt3 mutant exhibited a significant reduction in plant height (Figure S3). However, key yield components showed no significant differences, ultimately resulting in no measurable reduction in single-plant yield. These results demonstrate that emt3 is a promising target for optimising flowering time in rice without compromising yield. To further investigate the natural variation of the EMT3 gene, we performed a haplotype analysis using a diverse panel of 533 cultivated rice accessions, which classified the EMT3 into five major haplotypes: H1 was enriched in japonica (TeJ, TrJ), whereas H2-H5 predominated in indica (Aus, IndI, IndII) (Figure 1O). A key C-to-G SNP at position 1460 caused a proline-to-arginine substitution at position 487 of the encoded protein, showing clear indica-japonica differentiation (Figure 1O). The accessions carrying the EMT3G allele exhibited lower XGT activity and earlier flowering time than those with the EMT3C allele (Figure 1P,Q). We developed near-isogenic lines (NILs) by introgressing the japonica EMT3C allele into the indica II-32B background. NIL-EMT3C flowered approximately 0.5 h later than NIL-EMT3G (Figure 1R; Figure S8), suggesting that cumulative effects of multiple weak alleles contribute to flowering time divergence between indica and japonica. Investigation: P.X., T.W. and M.M.; formal analysis: K.D., H.Z., D.X. and J.W.; resources: Z.Z., X.L. and A.Z.; visualisation: M.M., T.W. and H.Z.; writing: P.X. and H.Z.; supervision: M.L., C.S., H.Z. and X.W.; funding acquisition: P.X., H.Z. and X.W.; field management: J.W. The data that supports the findings of this study is available in the Supporting Information of this article. Data S1: Supporting Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.