Disruption of mitochondrial open reading frame 352 partially restores pollen development in cytoplasmic male sterile rice.

Plant mitochondrial genomes sometimes carry cytoplasmic male sterility (CMS)-associated genes. These genes have been harnessed in various crops to produce high-yielding F1 hybrid seeds. The gene open reading frame 352 (orf352) was reported to be an RT102-type CMS gene in rice (Oryza sativa), although the mechanism underlying its role in CMS is unknown. Here, we employed mitochondrion-targeted transcription activator-like effector nucleases (mitoTALENs) to knockout orf352 from the mitochondrial genome in the CMS rice RT102A. We isolated 18 independent transformation events in RT102A that resulted in genome editing of orf352, including its complete removal from the mitochondrial genome in several plants. Sequence analysis around the mitoTALEN target sites revealed their induced double-strand breaks were repaired via homologous recombination. Near the 5'-target site, repair involved sequences identical to orf284, while repair of the 3'-target site yielded various new sequences that generated chimeric genes consisting of orf352 fragments. Plants with a chimeric mitochondrial gene encoding amino acids 179-352 of ORF352 exhibited the same shrunken pollen grain phenotype as RT102A, whereas plants either lacking orf352 or harboring a chimeric gene encoding amino acids 211-352 of ORF352 exhibited partial rescue of pollen viability and germination, although these plants failed to set seed. These results demonstrated that disruption of orf352 partially restored pollen development, indicating that amino acids 179-210 from ORF352 may contribute to pollen abortion.

Mammalian Bax initiates plant cell death through organelle destruction.

Mammalian Bax is known to cause cell death when expressed in plants. We examined transgenic plants expressing both Bax and organelle-targeted green fluorescent protein to determine the cellular changes that occur during Bax-induced cell death. The mitochondria changed morphologically from being bacilli-shaped to being round, eventually becoming swollen. Mitochondria streaming also stopped. The chloroplasts lost membrane function and their contents leaked out, followed by the disruption of the vacuole. Light was not essential for Bax-induced ion leakage or organelle disruption. These results indicate that Bax induces temporal and spatial cell death events at the organelle level in the plant. A heterologous system, using Bax, would therefore be available to investigate cell death, which is commonly conserved in animals and plants.