Nuclease activities associated with dark-induced and natural leaf senescence in parsley

Abstract

Leaf senescence is an endogenously controlled degenerative process leading to cell death. Multiple catabolic pathways induced during senescence are responsible for breakdown of different components of the cell. The hydrolytic enzymes involved play an important role in eliciting death. We used the dark-induced accelerated senescence of detached parsley leaves to study nucleases induced during the process. These enzymes are thought to play a role in the senescence-induced massive degradation of the cell's nucleic acid content. RNase and DNase activities whose levels increased in parallel with the advance of senescence were identified with the activity gel assay. We focused on characterization of two nucleases, PcNUC1 (∼43 kDa) and PcNUC2 (∼40 kDa) which were observed to be induced early in the dark incubation of detached leaves, and to increase steadily as senescence progressed. Both nucleases were found to be glycosylated and could degrade both RNA and DNA, with optimal activity under basic pH (8.5–9.0). EDTA inhibited the activity of both PcNUC1 and 2, while the addition of Co 2+ or Mn 2+ could restore their activity in the presence of the chelating agent. PcNUC1 and 2 activities in detached green leaves were enhanced by ethylene treatment and were found to be induced in attached parsley leaves undergoing natural senecence. Both activities can be classified as Nuclease I type enzymes and show high similarities to the previously described LeNUC1 nuclease which is induced during the natural senescence of tomato leaf. These nucleases may be involved in the massive nucleic acid catabolism process which occurs during leaf senescence.