Reduced expression of top1b gene induces programmed cell death and alters ascorbate metabolism in Daucus carota cultured cells

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 top1beta gene encoding the topo Ibeta 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.