Oxidative stress is generated via the mitochondrial respiratory chain during plant cell apoptosis.
BACKGROUND
We present evidence that in plant cells DNA damage induced by the topoisomerase trapping drug camptothecin induces oxidative stress via the mitochondrial respiratory chain.


METHODS
Flow cytometry was used to analyse mitochondrial respiratory chain activity by simultaneous measurement of mitochondrial generation of reactive oxygen intermediates (ROI) and mitochondrial membrane potential (DeltaPsim), in live functioning sugarbeet protoplasts. The mitochondrial respiratory chain function was assessed by the addition of substrate and inhibitors to the digitonin-permeabilised protoplasts. Mitochondrial activity was also measured in protoplasts at different stages of induced apoptosis by camptothecin.


RESULTS
The addition of camptothecin to sugarbeet protoplasts resulted in increased mitochondrial generation of ROI, occurring concurrently with the hyperpolarisation of the mitochondrial inner membrane. Subsequently, there was a decrease in mitochondrial-generated ROI, in association with a decrease in DeltaPsim. These camptothecin-treated digitonin-permeabilised protoplasts could be re-energised and ROI decreased by the addition of malic acid in the early stages of apoptosis induction, but not in the latter stages. There was a depolarisation of the mitochondrial inner membrane in the final stages of apoptosis.


CONCLUSIONS
These results are consistent with impaired adenine nucleotide exchange across the mitochondrial membrane, suggesting that similar mechanisms regulate plant apoptosis as those described for mammalian apoptosis.
Weir
Iona E
IE
Division of Experimental Therapeutics, Department of Medical Oncology and Haematology, Ontario Cancer Institute/Princess Margaret Hospital, Toronto, Canada. iweir@biodiscovery.co.nz
Pham
Nhu-An
NA
Hedley
David W
DW
D017209:Apoptosis; D002166:Camptothecin / Q000494:pharmacology; D004249:DNA Damage; D004072:Digitonin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004579:Electron Transport / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D007202:Indicators and Reagents / Q000494:pharmacology; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000473:pathology*; D018384:Oxidative Stress; D010101:Oxygen Consumption; D059828:Plant Cells; D017382:Reactive Oxygen Species; D013997:Time Factors
D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D017382:Reactive Oxygen Species; D004072:Digitonin; D002166:Camptothecin