Transient Expression of Mitochondrial-Targeted Peptides Linked to Cell Death Signaling and Pollen Collapse in S Male-Sterile Maize.

In maize, the S type of cytoplasmic male sterility (CMS-S) conditions pollen collapse at the early bi-cellular stage. A terminal deoxynucleotidyl transferase dUTP nick end labeling  (TUNEL) assay demonstrated nuclear and chromatin fragmentation in pre-collapse CMS-S pollen consistent with mitochondrial-signaled programmed cell death The main objective of this research is to identify the mitochondrial open reading frame (orf) responsible for these events. Mitochondrial transcripts are associated with CMS-S pollen collapse, but post-transcriptional RNA editing creates different versions of the orfs suspected to be responsible for pollen cell death. The up-stream frame (orf355) is not edited, but the down-stream frame (orf77) is edited to create two shorter orfs (orf17 and orf26). Furthermore, partial editing within orf17 creates S14 and L14 coding variants. Thus far, plant mitochondrial genomes cannot be genetically transformed, so a transient expression approach was taken. Candidate orfs were fused to a mitochondrial targeting sequence, the ATP9 double leader (ATP9DL) from Neurospora crassa, and expressed behind the 35S or ap3 promoter. Fluorescence microscopy of stable Arabidopsis transformants demonstrated  that ATP9DL targets GFP to mitochondria creating the potential to successfully target orf translation products. Transient experiments conducted by Agrobacterium infiltration will test whether mitochondrial-targeted peptides condition cell death lesions in tobacco leaves and petunia petals.