Fig. 1. A comprehensive schematic illustrating genomic organization and structural features of OsDOMON genes. A, Schematic representation of the chromosomal distribution of rice DOMON genes. The physical positions (cM) of OsDOMONs were mapped according to the rice genome. B, Gene structures illustrating the exon-intron organization of the DOMON genes in rice. CDS, Coding sequence. C, Conserved domain analysis of OsDOMONs was conducted using TBtools. This analysis revealed the conservation of the OsDOMON domain along with other conserved domains like the Cytochrome_b_N superfamily among the genes. Different domains are represented in different colors as indicated in the figure. D, Conserved motifs of OsDOMON peptides were investigated using MEME. Different conserved motifs are represented by different colored boxes, and the motif sequences are given at the bottom. The size of the boxes indicates the length of the conserved motifs.

Fig. 2. Phylogenetic analysis and synteny analysis of DOMON proteins in rice and other plant species. A, Phylogenetic relationship among different OsDOMONs. B, Phylogenetic relationship between different OsDOMONs in rice from three different cultivars HP2216 (HP), Nipponbare (NIP), and Tetep (Tet). OsDOMONs from HP2216 are marked in blue, Nipponbare in black, and Tetep in red. The tree was constructed based on the full-length sequences of proteins. C, Phylogenetic relationships of DOMON proteins from five different plant species were generated by MEGAX with 1000 bootstrap replicates using the maximum likelihood method. The DOMON genes from O. sativa are marked in maroon, yellow for Brachypodium distachyon, blue for Triticum aestivum, purple for Arabidopsis thaliana and black for Zea mays. D, Visualization of evolutionary relationship of DOMONs from four different plant species B. distachyon, T. aestivum, A. thaliana, and Z. mays using Circos plot.

Fig. 3. Superimposed AlphaFold-predicted 3D structures of representative OsDOMON proteins. Structural alignment of OsDOMON3.2, OsDOMON1.4, OsDOMON6.1, and OsDOMON8.1 illustrates the conserved β-sandwich fold characteristic of DOMON domains. Despite differences in the adjacent domain architecture, the core structural fold is preserved across all four models, indicating strong structural conservation within the family.

Fig. 4. Expression profiles of OsDOMON genes based on RNA-seq data in biotic and abiotic stresses. A, Heatmap displays the expression profiles of OsDOMON genes based on RNA-seq data of rice panicle tissues infected with Magnaporthe oryzae at 48, 72, and 96 h post-infection (hpi). The heatmap was constructed using TBtools. hpi, Hours post inoculation. B, Venn diagram representing the gene expression patterns of the OsDOMON genes in different biotic stresses using publicly available RNA-seq data from the RiceMetaSysB database. C, Gene expression patterns of the OsDOMON genes in different abiotic stresses using publicly available RNA-seq data from the RiceMetaSys and RiceMetaSysHRG databases.

Fig. 5. Expression analysis of OsDOMON genes in different tissues (leaf, stem, root, and panicle) at the panicle stage of Tetep and HP2216 rice cultivars using qRT-PCR. Gene expression was analyzed in different tissues, with leaf tissue taken as the control for relative expression. Ubiquitin was used as the internal control. Data are mean ± SE (n = 3). The lowercase letters above bars indicate significant differences at the 0.05 level by the least significant difference test.

Fig. 6. Expression analysis of OsDOMON genes of Tetep and HP2216 after Magnaporthe oryzae infection at 6, 12, 24, and 48 hours post-inoculation intervals using qRT-PCR. Gene expression was analyzed in leaf tissues at the seedling stage, relative to 0 post-inoculation (at the time of infection). Ubiquitin was used as the internal control. Data are mean ± SE (n = 3). The lowercase letters above bars indicate significant differences at the 0.05 level by the least significant difference test.

Fig. 7. Detection of reactive oxygen species accumulation by nitroblue tetrazolium (NBT A and B) staining and cell death by trypan blue staining (C and D) in rice leaves of Hp2216 (A and C) and Tetep (B and D) under abiotic stress conditions.

Fig. 8. Expression analysis of OsDOMON genes in Tetep and HP2216 under drought (A), heat (B), and salt (C) stresses using qRT-PCR. Gene expression was analyzed in leaf tissues, relative to unstressed leaf tissue. Ubiquitin was used as the internal control. Data are mean ± SE (n = 3). The lowercase letters above bars indicate significant differences at the 0.05 level by the least significant difference test.