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

A recent study indicated that development of lesions on 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 if the cells 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 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 cells from Zn-exposed leaves, together with enhanced expression of three PCD marker genes (NtBI-1, Ntrboh, and NtSIPK) indicated 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, at exposure to high Zn, in situ localization of NO (visualized by DAF-2DA fluorescence) was restricted to groups of mesophyll cells, and correlated with the pattern of Zn localization (determined by 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 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.