Aspects of programmed cell death during leaf senescence of mono- and dicotyledonous plants

Leaf senescence is a highly regulated stage in the plant life cycle, leadin g to cell death, recently examined as a type of the programmed cell death ( PCD). One of the basic features of PCD is the condensation of nuclear chrom atin which is caused by endonucleolytic degradation of nuclear DNA (nDNA). In our investigations, we applied the technique of the single-cell electrop horesis system ("comet assay") in order to determine the type of nDNA fragm entation during leaf senescence. The comet assay, a sensitive method reveal ing nonrandom internucleosomal damage that is specific for PCD, is especial ly useful for the detection of nDNA degradation in isolated viable cells. S imultaneously, we analyzed the mesophyll cell ultrastructure and the photos ynthetic-pigment concentration in the leaves of two species, Ornithogalum v irens and Nicotiana tabaccum, representing mono- and dicotyledonous plants which differ in the pattern of leaf differentiation. These investigations d emonstrated that, in both species, the comet assay revealed nDNA degradatio n in yellow-leaf protoplasts containing chloroplasts that showed already ch anged ultrastructure (swelled or completely degraded thylakoids) and cell n uclei with a significant condensation of chromatin. There was no nDNA degra dation in green-leaf protoplasts containing differentiated chloroplasts wit h numerous grana stacks and nuclei with dispersed chromatin. The analysis o f intermediate developmental stage showed that the degradation of nDNA prec edes condensation of nuclear chromatin. Thus the comet assay is a very usef ul and sensitive method for early detection of PCD. Moreover, results of ou r studies indicate that leaf senescence involves PCD.