Fig. 4: Fertilization-induced autocrine ZmRALF12 signal mediates mitochondrial oxidative stress and synergid apoptosis.
From: Fertilization-induced synergid cell death by RALF12-triggered ROS production and ethylene signaling
a 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 (4 μM for 30 min) 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, 20 μm (a) and 10 μm (b, d, h–k), respectively.
