Kinetics and localization of wound-induced DNA biosynthesis in potato tuber.

Tuber wounding induces a cascade of biological responses that are involved in processes required to heal and protect surviving plant tissues. Little is known about the coordination of these processes, including essential wound-induced DNA synthesis, yet they play critical roles in maintaining marketability of the harvested crop and tubers cut for seed. A sensitive "Click-iT EdU Assay" employing incorporation of the thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU), in conjunction with 4',6-diamindino-2-phenylindole (DAPI) counter labeling, was employed to objectively identify and determine the time course and spatial distribution of tuber nuclei that were wound-induced to enter S-phase of the cell cycle. Both labeling procedures are rapid and sensitive in situ. Following wounding, EdU incorporation (indicating DNA synthesis) was not detectable until after 12h, rapidly reached a maximum at about 18h and then declined to near zero at 48h. About 28% of the nuclei were EdU labeled at 18h reflecting the proportion of cells in S-phase of the cell cycle. During the ∼30h in which induced cells were progressing through S-phase, de novo DNA synthesis extended 7-8 cell layers below the wound surface. Cessation of nuclear DNA synthesis occurred about 4 d prior to completion of wound closing layer formation. Initiation of wound periderm development followed at 7 d, i.e. about 5 d after cessation of nuclear DNA biosynthesis; at this time the phellogen developed and meristematic activity was detected via the production of new phellem cells. Collectively, these results provide new insight into the coordination of wound-induced nucleic acid synthesis with associated tuber wound-healing processes.