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First published online March 10, 2006; 10.1104/pp.106.078683

Plant Physiology 141:208-219 (2006)
© 2006 American Society of Plant Biologists

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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Cytochrome c Is Released in a Reactive Oxygen Species-Dependent Manner and Is Degraded via Caspase-Like Proteases in Tobacco Bright-Yellow 2 Cells en Route to Heat Shock-Induced Cell Death1

Rosa Anna Vacca, Daniela Valenti, Antonella Bobba, Riccardo Sandro Merafina, Salvatore Passarella and Ersilia Marra*

Istituto di Biomembrane e Bioenergetica, Consiglio Nazionale delle Ricerche, I–70126 Bari, Italy (R.A.V., D.V., A.B., R.S.M., E.M.); and Dipartimento di Scienze per la Salute, Università del Molise, I–86100 Campobasso, Italy (S.P.)

To gain some insight into the mechanism of plant programmed cell death, certain features of cytochrome c (cyt c) release were investigated in heat-shocked tobacco (Nicotiana tabacum) Bright-Yellow 2 cells in the 2- to 6-h time range. We found that 2 h after heat shock, cyt c is released from intact mitochondria into the cytoplasm as a functionally active protein. Such a release did not occur in the presence of superoxide anion dismutase and catalase, thus showing that it depends on reactive oxygen species (ROS). Interestingly, ROS production due to xanthine plus xanthine oxidase results in cyt c release in sister control cultures. Maximal cyt c release was found 2 h after heat shock; later, activation of caspase-3-like protease was found to increase with time. Activation of this protease did not occur in the presence of ROS scavenger enzymes. The released cyt c was found to be progressively degraded in a manner prevented by either the broad-range caspase inhibitor (zVAD-fmk) or the specific inhibitor of caspase-3 (AC-DEVD-CHO), which have no effect on cyt c release. In the presence of these inhibitors, a significant increase in survival of the cells undergoing programmed cell death was found. We conclude that ROS can trigger release of cyt c, but do not cause cell death, which requires caspase-like activation.

1 This work was supported by the Italian Ministry of Instruction, University and Research, "Contributi straordinari di ricerca/aree obiettivo 1" (to E.M.) and by Programmi di Ricerca a Rilevante interesse Nazionale, "I mitocondri nello stress e nella morte cellulare in sistemi vegetali" (to S.P.).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ersilia Marra (e.marra{at}ibbe.cnr.it).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.078683.

* Corresponding author; e-mail e.marra{at}ibbe.cnr.it; fax 39–080–5443317.

Received February 3, 2006; returned for revision February 27, 2006; accepted February 27, 2006.




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