ZmRALF12 application assay using an optimized ovule cultivation system. Sectioned ovules are double-stained with DCFH-DA and Mito-Tracker Red to visualize the ZmRALF12 response and mitochondria localization. ZmRALF1 and ZmRALF2 application were negative (Supplementary Fig. 7 ). ZmRALF12 application (4M for 30min) triggers high levels of mitochondrial ROS accumulation at the FAAR in manually isolated synergid cells ( b , c ), comparable to persistent synergid cells isolated at 20 HAP ( d , e ). Asterisks indicate moderate ROS accumulation in synergid nuclei. Merged images of ROS accumulation and a mito-tracker are as indicated ( b , d ). Intensity plots ( c , e ) were generated along the green arrows in ( b , d ). f In contrast to the activation of extracellular targeted ROS scavenging enzymes, mitochondrial ROS scavenging enzymes are downregulated after fertilization and continually decrease during synergid degeneration. g Mitophagy and senescence-related genes are strongly activated at 24 HAP. h , i Monodansylcadaverine (MDC) staining showing an intact synergid cell before fertilization ( h ) and active autophagic processes and vacuole accumulation in the persistent synergid cell before its elimination ( i ). j , k ZmRALF12, but not ZmRALF2, can effectively trigger autophagosome production and vacuole accumulation in synergid cells, comparable to persistent synergid cells at 24 HAP ( i ). Green arrows point to autophagosomes transported to vacuoles. The white arrow shows autophagy maxima at the mitochondrial ROS accumulating FAAR. ZmRALF12 application assays ( a , b ), DCFH-DA and Mito-Tracker Red double staining of persistent synergid cells at 20 HAP ( d ), MDC staining of synergid cells at 0 HAP ( h ) and persistent synergid cells at 24 HAP ( i ), as well as MDC staining after ZmRALF12 ( j ) or ZmRALF2 ( k ) treatment were repeated three times with similar results. FA filiform apparatus, FAAR filiform apparatus adjacent region, SC synergid cell. Scale bars, 20m ( a ) and 10m ( b , d , h – k ), respectively.