In Situ Detection of Programmed Cell Death in Senescing Nicotiana tabacum Leaves Using TUNEL Assay
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© Springer Science+Business Media, LLC 2018
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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.
Keywords:DNA fragmentation; Histology; Leaf senescence; Nicotiana tabacum; Programmed cell death; TUNEL assay
In: Guo Y, editor. Plant Senescence: Methods and Protocols. New York, NY: Humana Press; 2018. p. 267–82. (Methods in Molecular Biology; Vol. 1744)