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JXB Advance Access published online on May 22, 2006
Journal of Experimental Botany, doi:10.1093/jxb/erj194
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© The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org
Received January 24, 2006
Accepted March 13, 2006

Oxygen Metabolism Special Issue

Reduced expression of top1{beta} gene induces programmed cell death and alters ascorbate metabolism in Daucus carota cultured cells

Vittoria Locato 1 *, Alma Balestrazzi 2 *, Laura De Gara 3 *, and Daniela Carbonera 2

1 Department of Plant Biology and Pathology, University of Bari, Via Orabona 4, I-70125, Bari, Italy
2 Department of Genetics and Microbiology, University of Pavia, Via Ferrata 1, I-27100, Pavia, Italy
3 Department of Plant Biology and Pathology, University of Bari, Via Orabona 4, I-70125, Bari, Italy; Interdisciplinary Center for Biomedical Research (CIR) Università Campus Biomedico Via Longoni 83, I-00155 Roma, Italy

* To whom correspondence should be addressed.
Laura De Gara, E-mail: degara{at}botanica.uniba.it


  Abstract

Topoisomerase I (topo I) is a nuclear enzyme which plays a fundamental role in several pathways involving changes in DNA topology. The topo I-mediated reaction is accomplished by the transient covalent binding of the enzyme to DNA (topo I-DNA complex). Stabilization of the topo I-DNA complex, leading to irreversible double-strand breaks, has been reported to occur in animal cells under oxidative stress conditions and during apoptosis. In order to study the existence of a putative link between the topo I-mediated DNA damage and ascorbate (ASC) metabolism, also involved in the responses against oxidative stress and in the apoptotic process in plants, Daucus carota cells showing reduced expression of the top1{beta} gene encoding the topo I{beta} isoform were produced, using an antisense RNA strategy. Two independent transgenic lines (AT1-{beta}/22 and {beta}/36), characterized by a slow growth phenotype, resistance to camptothecin, a specific inhibitor of topo I, but sensitivity to etoposide, an inhibitor of topo II, were investigated in this study. In the absence of external stimuli, AT1-{beta}/22 and {beta}/36 cells underwent programmed cell death (PCD) in a precocious phase of the growth curve. ASC metabolism showed remarkable differences in AT1-{beta}/22 and {beta}/36 cells, compared with control, and the observed alterations were similar to those occurring in tobacco Yellow Bright-2 cells induced to enter PCD by exogenous stimuli. However, differently from other studied examples of PCD, overproduction of reactive oxygen species was not detected in AT1-{beta}/22 and {beta}/36 cells. The relevance of these findings in relation to the signalling pathways leading to PCD is discussed.

Keywords: Antioxidants; ascorbate; Daucus carota; programmed cell death; topoisomerase.

*These authors contributed equally to the present work.


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