<scp><i>OsFON879</i></scp>, an orphan gene, regulates floral organ homeostasis in rice

Floral organ development in angiosperms is tightly regulated by conserved pathways, including the ABCE model and CLV-WUS signalling, which coordinate meristem activity and organ identity (Litt and Kramer, 2010; Schoof et al., 2000). However, the mechanisms underlying species-specific floral architecture, particularly in monocots like rice (Oryza sativa), remain poorly understood. Although key regulators such as FON1/FON2 and ABCE-class genes coordinate floral meristem maintenance (Hu et al., 2015; Ren et al., 2019; Suzaki et al., 2004), the contribution of lineage-specific genetic innovations, particularly orphan genes (OGs), to floral organ development remains poorly characterized. OGs, which lack detectable homologues or conserved domains across species, have emerged as key contributors to species-specific traits and adaptive evolution (Tautz and Domazet-Lošo, 2011). Despite their importance, OGs remain understudied due to challenges in functional characterization. Recent studies have implicated OGs in specialized developmental processes (Jiang et al., 2022), but their precise roles in plant development are not well understood. Here, we characterize OsFON879 (LOC_Os03g12879), a rice-specific OG encoding a predicted intrinsically disordered protein (IDPs) based on AlphaFold (Figures S1–S3). IDPs are known to act as dynamic scaffolds, facilitating interactions between multiple macromolecules (e.g., proteins, RNA) in regulatory hubs (Chakrabarti and Chakravarty, 2022). In situ hybridization (Figure 1a1–a6) and RT-qPCR assay (Figure 1a7) revealed OsFON879 expression specifically in reproductive tissues, peaking during early panicle development. These results indicate that the IDP of OsFON879 may play an important role in the floral development process of rice. CRISPR-Cas9-induced mutant osfon879 exhibited stochastic floral organ defects and a moderate increase in tiller number during panicle development, which were fully rescued in genetic complementation lines (Figure 1b1–b4). Twenty-four per cent of the osfon879 mutant flowers had fewer stamens (minimum of four per flower), and 30% had more (up to nine per flower). Also, 70% of mutant flowers had more stigma (up to nine per flower). The carpels were affected too, with 32% of flowers having two and 12% having three. A few spikelets had more glumes (Figure 1c1–c7). The stochastic floral organ patterning in osfon879 mutants implicates a role in maintaining meristem homeostasis; however, its lack of conserved protein domains implies a novel regulatory mechanism diverging from canonical pathways. In situ hybridization demonstrated suppressed OsMADS1 expression in osfon879 mutants (Figure 1d1–d8). Given that OsMADS1, an E-class MADS-box gene, is critical for floral organ identity specification (Je et al., 2016), this implies OsFON879 modulates OsMADS1 expression. The subcellular localization results indicate that OsFON879 is predominantly localized in the nucleus, with smaller amounts present in the cytoplasm and cell membrane (Figure 1e1). Yeast two-hybrid screening revealed that OsRRM1, an RNA-binding protein, interacts with OsFON879 (Figure 1e2). The OsFON879-OsRRM1 interaction was independently confirmed through co-immunoprecipitation (Co-IP) in rice 7-day root protoplast systems (Figure 1e3) and bimolecular fluorescence complementation (BiFC) assays in Nicotiana benthamiana epidermal cells (Figure 1e4), definitively confirming their direct physical association. In osfon879 mutants, the expression of OsRRM1 was significantly decreased (Figure 1f1). Furthermore, RT-qPCR (Figure 1f2) and in situ hybridization analyses (Figure 1g1–g7) revealed a spatiotemporal co-expression pattern of OsRRM1 and OsFON879, with both genes exhibiting high and tissue-specific expression in rice floral organs. These findings reveal that OsFON879 directly regulates the expression of OsRRM1, highlighting their functional interdependence.OsRRM1 contains an RNA recognition motif (RRM) which is predicted to bind mRNAs encoding floral meristem regulators (e.g., OsMADS1 and OsWUS) based on RiceFREND database analysis (Sato et al., 2013). The interaction and spatiotemporal coexpression of OsFON879 and OsRRM1 in floral organs, combined with reduced OsRRM1 expression in osfon879 mutants, supports a model where OsFON879 stabilizes OsRRM1 activity. We propose that OsFON879 recruits OsRRM1 to modulate RNA stability or translation efficiency of target transcripts, thereby regulating meristem activity. This hypothesis is consistent with the stochastic floral organ phenotypes observed in osfon879 mutants, which reflect disrupted RNA processing. Importantly, the regulatory effect of OsFON879 on OsMADS1 expression level in the osfon879 mutant identifies OsFON879 as a central player in floral organogenesis. We propose that the OsFON879-OsRRM1 interaction stabilizes OsMADS1 transcripts, ensuring robust E-class function during floral organ specification. This study identifies OsFON879 as a rice-specific OG critical for floral organ development regulation. OsFON879's orphan status and lack of conserved domains suggest a novel regulatory layer. The interaction with OsRRM1 links orphan genes to RNA metabolism, expanding known floral meristem networks beyond transcriptional control. Our findings highlight orphan genes as untapped resources for engineering agronomic traits and advance understanding of post-transcriptional regulation in plant development. Future studies should explore OsFON879-OsRRM1's role in transcriptome-wide RNA processing and their genetic interactions with known meristem regulators. We thank Prof. Fan Chen (Institute of Genetics and Developmental Biology, CAS) for yeast two-hybrid technical support. National Natural Science Foundation of China (31630006). The authors declare no competing interests. D.H.W. designed and performed experiments and wrote the manuscript. Z.H.X. and S.N.B. supervised and revised the manuscript. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Figure S1 AlphaFold structural prediction of OsFON879. Highlighted regions: Low-confidence/disordered segments (yellow) and very low-confidence segments (orange) (https://alphafoldserver.com/). Figure S2 Showing features for region of OsFON879 with UniProt feature annotations. Highlighted regions: Disordered segments (orange) (https://www.uniprot.org/uniprotkb/Q10PL1/entry). Figure S3 Sequences producing significant alignments with NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Table S1. Target sequences used for CRISPR/Cas9-mediated gene knockout. Table S2. Primer pairs used in this study. Data S2. Materials and Methods. 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.