Fig. 3: ROS production in the persisting synergid cell is correlated with fertilization-induced accumulation of ZmRALF12 at the filiform apparatus.

a DCFH-DA staining showing ROS accumulation in epidermal micropylar nucellus cells and its absence inside the mature embryo sac. b, c At 20 HAP strong staining is detectable inside the persistent synergid cell. White arrowheads point to ROS maxima in persistent synergid cells. d Quantification of ROS fluorescence intensity in synergid cells at stages shown in (a–c) (n = 10). e, f DCFH-DA and Mito-Tracker Red double-staining showing mitochondria constitutively accumulating alongside the filiform apparatus (FA) adjacent region (FAAR) in synergid cells (black arrowheads). ROS are undetectable in synergid cells before pollination (e) and accumulate at the mitochondria-enriched FAAR in the persistent synergid cell at 20 HAP (f). g, h Expression of RBOH and RALF peptide/receptor genes during fertilization and persistent synergid degeneration. Transcript levels are shown as FPKM values (means ± SD) of three biological replicates. For statistical analysis, count data at the gene level were analyzed with DESeq2⁶⁴. Stage-to-stage comparisons were performed and corrected for multiple testing over all genes and cell stage comparisons using false discovery rate (FDR). Letters above bars indicate significant differences (adjusted P < 0.05). i–l Immunostaining showing secreted ZmRALF12 peptides at the FA from 18 HAP (k) and increasing amounts at 24 HAP at both, FA and FAAR regions. m DCFH-DA and Mito-Tracker Red double-staining in zmralf12 mutant embryo sacs. ROS are not induced at the mitochondria-accumulating FAAR in the persistent synergid cell at 20 HAP. In box-and-whisker plots, center lines represent the 50th percentile; bottom and top of each box indicate the 25th and 75th percentiles, respectively; whiskers represent minimum and maximum, respectively. Statistical significance is determined by two-tailed Student’s t-test. Source data and adjusted P values (in g, h) are provided in the source data file. DCFH-DA and Mito-Tracker Red double-staining of intact synergid cells in embryo sacs 0 HAP (e) and 20 HAP (f), as well as ZmRALF12 immunostaining (i–l), and DCFH-DA and Mito-Tracker Red double-staining of zmralf12 mutant embryo sacs 20 HAP (m) were repeated three times with similar results. dSC degenerated receptive synergid cell, FA filiform apparatus, pSC persistent synergid cell, SC synergid cell. Scale bars: 50 μm.