NO-Dependent programmed cell death is involved in the formation of Zn-related lesions in tobacco leaves

Abstract

A recent study indicated that the development of lesions on the leaf blades of tobacco exposed to zinc (Zn) excess can be considered a manifestation of a Zn-tolerance strategy at the organ level. Here, we investigated whether cell death leading to the formation of localized lesions is destructive in character (necrosis type) or results from programmed self-induced cell death (PCD). Selected parameters, including PCD markers, were determined in the leaves from tobacco plants grown in the presence of 200 μM Zn and compared with control conditions. TUNEL assay results showing internucleosomal DNA fragmentation in the nuclei of the cells from Zn-exposed leaves, together with an enhanced expression of three PCD marker genes (NtBI-1, Ntrboh, and NtSIPK), indicated the involvement of PCD in the formation of Zn-related lesions. It is known that NO is a key factor in the execution of PCD. Interestingly, upon exposure to high Zn, in situ localization of NO (visualized using DAF-2DA fluorescence) was restricted to groups of mesophyll cells, and was correlated with the pattern of Zn localization (determined using the fluorophore Zinpyr-1), similarly limited primarily to groups of “Zn accumulating cells”. Furthermore, inhibition of the formation of lesions in the presence of L-NAME (an NO synthase inhibitor) was accompanied by the delayed appearance of Zn and by NO localization limited to these groups of cells. Altogether, we provide the first demonstration that Zn-related lesions in leaves develop from groups of mesophyll cells in which accumulation of high concentrations of Zn contributes to enhancement of the NO level and to initiation of PCD processes.