Mitochondrial involvement in plant programmed cell death

Programmed cell death (PCD) is a genetically controlled pathway for regulated cell destruction. It is fundamentally important for plant development and mediates defense strategies against abiotic and biotic stimuli. However, compared to the well characterized cell death programs in animals, our understanding of PCD in plants and its regulatory components is less advanced. The mitochondrion plays a central signaling role in many programmed cell death subroutines described in animals, including mammalian apoptosis. There is considerable experimental evidence for mitochondrial involvement in PCD regulation to also be conserved in plants, but details of how mitochondria contribute to plant PCD signaling require further investigation. This thesis provides new insights into mitochondria-PCD cross-talk in plants by exploring two different aspects of mitochondrial regulation of cell death signaling. The release of mitochondrial intermembrane space proteins such as cytochrome c (cyt c) into the cytosol is a key event during apoptosis leading to activation of cell death signaling cascade. The first research chapter of this thesis aimed to identify proteins that undergo similar translocation upon induction of PCD in plants. To achieve that a well-established system for studying plant PCD, Arabidopsis thaliana cell suspension culture, was used in combination with a cellular fractionation protocol and mass spectrometry-based proteomics to examine changes in protein abundance and localization associated with the early stages of heat stress-induced PCD. This experimental set up facilitated characterization of rapid and extensive changes in protein abundance in both mitochondrial and cytosolic fraction, and identification of 113 mitochondrial proteins that may undergo release into cytosol upon PCD induction and therefore are interesting targets of future research. Functional enrichment analyses highlighted the role pathways associated with stress response, proteasome function, protein folding, and desoxyribonucleic acid (DNA) repair in response to applied heat stress and potentially also in PCD regulation. Further, the PCD-specific release of mitochondrial HSP60 was demonstrated, underlining its possible role in PCD regulation in plants. The second research chapter of this thesis is focused on the role of mitochondrion as the organelle sensing and integrating stress responses in plant cells by exploring the cross talk between PCD and mitochondrial retrograde signaling (MRS), a pathway by which signals originating from mitochondria influences nuclear gene expression. The root hair assay, a method facilitating quantitative determination of PCD induced by a diversity of stimuli, and its modulation by pharmacological and genetic treatments, was employed to achieve this. The experiments combining PCD inducing stimuli with the chemical activator of MRS, Antimycin A (AA), and examination of the cell death response in the knockout line of the master regulator of MRS, transcription factor ANAC017, revealed that the MRS appears to function primarily as a pro-survival, PCD suppressing mechanism. However, phenotyping of available knockout/overexpression lines of the individual members of MRS regulome highlighted the complexity of this signaling pathway that requires further investigation.
Collectively, the work presented in this thesis underscores the mitochondrion as an organelle central to the regulation of decisions for cell death and survival in plants and will feed into future research work in this area.