Fig. 5: Fertilization-induced ROS and ethylene production act synergistically to promote synergid cell death in maize.

a–c Post-fertilization ROS scavenger application delays persistent synergid cell death/elimination. Ovules were harvested at 16 HAP and cultivated in an optimized culture medium (a; control) and medium supplied with the ROS scavenger CuCl₂ (5 mM, b), respectively. In contrast to control (a), ROS was almost non-detectable in persistent synergid cells after CuCl₂ application 22 HAP (b). c Frequency of persistent synergid cell-containing embryo sacs analyzed at 28 HAP (control: n = 28; +CuCl₂: n = 30). d–h Post-fertilization ethylene production activates the ethylene signaling pathway and promotes synergid cell death in maize. d Strong activation of ethylene (ET) biosynthesis and ET-responsive genes at 24 HAP in the persistent synergid cell. 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). For ET biosynthesis genes, significant differential expressions (DEs) are shown (*log₂FC > 1 and adjusted P < 0.05, ***log₂FC > 1 and adjusted P < 0.001). ET application specifically triggers only synergid cell death in the mature female gametophyte. CLSM of a female gametophyte without ET treatment (e; control) and 30 h after ET treatment exhibiting fully degenerated synergid cells (f) and degenerated synergid nuclei (g), respectively. h Frequencies of synergid defects (control: n = 119; +ET: n = 163). Statistical significance (in c, h) is determined by two-tailed Student’s t-test, and data are presented as mean values ± SD. Source data and adjusted P values (in d) are provided in the source data file. CC central cell, CCN central cell nucleus, dSC degenerated receptive synergid cell by pollen tube reception, DSC degenerated synergid cell by ET treatment, DSN degenerated synergid nucleus by ET treatment, EC egg cell, ECN egg cell nucleus, EN endosperm, pSC persistent synergid cell, SC synergid cell, SCN synergid cell nucleus. Scale bars, 50 μm.