Fig. 6: Programmed cell death (PCD) of the persistent synergid cell is activated after successful double fertilization.

CLSM images of embryo sacs before pollination (a) and at 24 HAP (b) and 25 HAP (c), respectively. White arrowhead shows synergid cell nucleus. Black arrowheads point towards degenerating persistent synergid nuclei. White arrows indicate cell boundaries. d–g Fluorescence microscopy to monitor the process of persistent synergid elimination that occurs at about 26–30 HAP. Embryo sacs at indicated time points expressing ES4-GFP and SC1-GFP driven by their endogenous promoters, respectively. The diffusion of GFP signals from persisting synergid cells to the endosperm was observed at 26 HAP (g). h Phylogenetic tree of NAC family PCD transcription factors from Arabidopsis and their maize homologs expressed during synergid degeneration. i, j Genes encoding a battery of PCD transcription factors with killing activity and their downstream PCD executors are activated/de novo expressed at 18–24 HAP. k Nicotiana benthamiana leaves infiltrated with NAC family PCD transcription factors that are significantly induced in persisting synergid cells at 24 HAP. l Activation patterns of selected maize genes encoding homologs of Arabidopsis ABA receptor, CDK inhibitor, and senescence regulators involved in cell death regulation. The two most strongly induced ncRNAs with similar activation patterns are shown. m De novo expressed unknown genes in the persistent synergid cell showing a PCD-related expression pattern. Data (in i, j, l) are presented as mean values ± SD. 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 indicate significant differences between developmental stages (adjusted P < 0.05). Source data and adjusted P values (i, j, l) are provided in the source data file. In (a–g, k) experiments were repeated three times with similar results and presented are representative images. CC central cell, dSC degenerated receptive synergid cell, EC egg cell, EN endosperm, pSC persistent synergid cell, PSN persistent synergid nucleus, SC synergid cell, SCN synergid cell nucleus, ZY zygote. Scale bars: 50 μm (a–g) and 2 cm (k), respectively.