In Situ Detection of Programmed Cell Death in Senescing Nicotiana tabacum Leaves Using TUNEL Assay

Abstract

Leaf senescence constitutes a highly regulated final phase of leaf development, leading to cell death that is recognized as a type of programmed cell death (PCD). Degradation of nuclear DNA into oligonucleosomal fragments (DNA ladder) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay are methods commonly used to detect PCD-specific DNA cleavage. TUNEL reaction in situ labels free 3′-OH DNA strand breaks (nicks), thus allowing histological localization of nuclear DNA degradation during PCD. Here we describe in situ labeling of PCD-specific nuclear DNA fragmentation on conventional histological sections of senescing tobacco leaves. Incorporation of fluorescein-labeled dUTPs is detected by fluorescence microscopy, which enables in situ visualization of PCD at the single-cell level in the leaf mesophyll tissues undergoing senescence.