A SIGNAL GLYCOPROTEIN WITH α-d-MANNOSYL RESIDUES IS INVOLVED IN THE WOUND-HEALING RESPONSE OF ANTITHAMNION SPARSUM (CERAMIALES,RHODOPHYTA)1

A variety of fluorescein isothiocyanate-labeled lectins specific for different sugar moieties were examined as probes for the wound-healing response in the filamentous red alga Antithamnion sparsum Tokida. Among them, only concanavalin A (ConA) and Lens culrinaris agglutinin (LCA), which have specificity to α-D-mannosyl residues, bound specifically to repair cells during the wound-healing process. When ConA or LCA was added at various time intervals after wounding, it first bound (3 h post-wounding) as a thin layer at the tips of the adjacent cells. Later (4–5 h post-wounding) labeling also appeared at the tips of the repair cells. Intense labeling at these sites continued throughout the healing process until repair cell fusion, at which time the lectin labeling was reduced to a narrow ring around the area of fusion. When added to plants prior to wounding and continually monitored, these same lectins acted as inhibitors to the wound-healing response. Other control lectins showed no inhibitory effects. A crude extract solution obtained from decapitated filaments stimulated the wound-healing response, and a lectin-binding component bound strongly to a protein-binding transfer membrane. These results suggest that the labeled compound is a glycoprotein that has α-D-mannosyl residues and is similar to the repair hormone rhodomorphin found in Griffithsia pacifica Kylin.     

Possible surface carbohydrates involved in signaling during conjugation process in Zygnema cruciatum monitored with fluorescein isothiocyanate-lectins (Zygnemataceae, Chlorophyta)

The changes of cell surface carbohydrates were examined with FITC (fluorescein isothiocyanate)‐labeled lectins during the conjugation process of the green alga Zygnema cruciatum. The Ulex europaeus agglutinin (UEA)‐specific materials were detected consistently on the surface of vegetative cells, but were absent on the surface of protruding papillae or conjugation tube. The tips of male and female papillae were labeled with soybean agglutinin (SBA) and peanut agglutinin (PNA) during conjugation. The SBA‐ and PNA‐specific materials appeared first at the tip of male papillae and began to accumulate on the surface of female papillae. No labeling of these lectins was detected on the surface of vegetative filaments throughout the conjugation process. FITC‐ConA (Concanavalin A) and FITC‐RCA (Ricinus communis agglutinin) did not label the vegetative filaments of Z. cruciatum, but a trace labeling of these lectins was observed on the surface of some swollen papillae occasionally. Blocking experiments with various lectins showed that these SBA‐ and PNA‐specific glycoconjugates might be involved in the signaling between male and female papillae.     

Binding of fluorescent lectins to the surface of germ cells from fetal and early postnatal mouse gonads

Fluorescent lectins were used to study the chemical nature of carbohydrate moieties present on the surface of female and male germ cells isolated from mouse gonads during fetal and early posnatal development. Concanavalin A (ConA), lens culinaris agglutinin (LCA), ricinus communis agglutinin (RCA_I) and wheat germ agglutinin (WGA) bound intensely to the germ cell plasma membrane at all stages studied. Other lectins such as ulex europaeus agglutinin (UEA_I) and agglutinin (SBA) did not bind or bound moderately (SBA to female germ cells only). Distinct developmental-related changes were observed when female germ cells were labeled with fluorescein-conjugated peanut agglutinin (PNA) or dolichos biflorus agglutinin (DBA). DBA and PNA binding was absent or weak in fetal female and male germ cells, but became intensely positive in oocytes in the immediate postnatal period. The percentage of oocytes stained with DBA increased during the first three days after birth, and from day 3–4 onwards all oocytes were strongly labeled. I suggest that these changes in lectin binding reflect changes in biochemical structure of the oocyte surface related to differentiative events occurring in the mouse ovary immediately after birth.     

Properties of cell surface carbohydrates in sexual reproduction of the Closterium peracerosum–strigosum–littorale complex (Zygnematophyceae,Charophyta)

The Closterium peracerosum–strigosum–littorale complex is a best characterized zygnematophycean green alga with respect to the process of sexual reproduction. Intercellular communication mediated by two sex pheromones has been well documented in this organism, but information concerning direct cell–cell recognition and fusion of cells involved in conjugation processes has not yet been clarified. In this study, we examined the properties of cell surface carbohydrates in vegetative and reproductive cells using a variety of fluorescein isothiocyanate labeled lectins as probes. Among 20 lectins tested, 10 bound to the Closterium cell surface, and eight of these were specific for the cells involved in sexual reproduction. In addition, some of the lectins inhibited the progress of zygote formation. In particular, Lycopersicon esculentum lectin (LEL) and ConcanavalinA (ConA) considerably inhibited zygote formation (23.6% and 0% of zygotes formed, respectively, compared with the control). LEL mainly accumulated on conjugation papillae and on the surface and lumens of empty cell walls remaining after zygote formation. ConA bound to both vegetative and sexually reproductive cells and strongly accumulated on the conjugation papillae of the latter, indicating ConA binding material(s) are non‐specifically present in Closterium cells but some of the material(s) would be essential for zygote formation. These results suggest that different carbohydrates specifically recognized by these lectins are involved in cell recognition and/or fusion during conjugation processes in the C. psl. complex.     

Characterization of the isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes by lectin cytochemistry

The isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes were characterized by lectin cytochemistry using concanavalin A (ConA), Ricinus communis agglutinin 120 (RCA-120), and wheat germ agglutinin (WGA). We found that RCA-120, ConA, and WGA bind to these membranes. The distribution of the lectins on the isolation membranes was heterogeneous, mainly found on the rims, which we referred to as the peripheral dilated portion. When the rims fused and thus formed autophagosomes the apparent sites of fusion were strongly labeled by the lectins. After autophagosomes were transformed to autolysosomes by fusion with lysosomes, the limiting membranes became more densely and homogeneously labeled with the lectins. We previously reported that cytochrome P-450 does not exist on the limiting membranes of the autophagosomes. Taken together, these results suggest that the isolation membranes may originate not from endoplasmic reticulum membranes but from some post-Golgi membranes that contain complex type N and/or O-linked oligosaccharide chains.     

Glycoconjugates in normal human kidney

Summary The avidin-biotin-peroxidase complex technique was used with 13 lectins to study the glycoconjugates of normal human renal tissue. The evaluated lectins included Triticum vulgaris (WGA), Concanavalin ensiformis (ConA), Phaseolus vulgaris leukoagglutinin and erythroagglutinin (PHA-L and PHA-E), Lens culinaris (LCA), Pisum sativum (PSA), Dolichos biflorus (DBA), Glycine max (SBA), Bandeiraea simplicifolia I (BSL-I), Ulex europaeus I (UEA-I) and Ricinus communis I (RCA-I). Characteristic and reproducible staining patterns were observed. WGA and ConA stained all tubules; PHA-L, PHA-E, LCA, PSA stained predominantly proximal tubules; DBA, SBA, PNA, SJA and BSL-I stained predominantly distal portions of nephrons. In glomeruli, WGA and PHA-L stained predominantly visceral epithelial cells; ConA stained predominantly basement membranes and UEA-I stained exclusively endothelial cells. UEA-I also stained endothelial cells of other blood vessels and medullary collecting ducts. Sialidase treatment before staining caused marked changes of the binding patterns of several lectins including a focal loss of glomerular and tubular staining by WGA; an acquired staining of endothelium by PNA and SBA; and of glomeruli by PNA, SBA, PHA-E, LCA, PSA and RCA-I. The known saccharide specificities and binding patterns of the lectins employed in this study allowed some conclusions about the nature and the distribution of the sugar residues in the oligosaccharide chains of renal glycoconjugates. The technique used in this report may be applicable to other studies such as evaluation of normal renal maturation, classification of renal cysts and pathogenesis of nephrotic syndrome. The observations herein reported may serve as a reference for these studies.     

Cell type-specific binding of Ricinus lectin to murine cerebellar cell surfaces in vitro

Summary The binding of several plant lectins, Concanavalin A (ConA), Lens culinarisA (LCA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin 120 (RCA120) to cell surfaces of developing mouse cerebellar cells was assayed by the use of fluorescein isothiocyanate (FITC)-conjugated compounds. Freshly dissociated, live single-cell suspensions from 6-day-old mouse cerebellum contain 93% ConA, 99% LCA, 98% WGA, and 59% RCA 120-positive cells with ring fluorescence. Of the RCA 120-positive cells, 4% express a high and 55% a lower or very low number of lectin receptors. Flow cytometric analysis of fluorescent lectin binding yields results qualitatively similar to those obtained by scoring positive and negative cells in the fluorescence microscope.In monolayer cultures of 6-day-old mouse cerebellum practically all cells express receptors for ConA, LCA, and WGA, whereas RCA 120 binding sites are absent from neurons with small cell bodies (granule, basket and stellate cells) and present in large number on neurons with large cell bodies (Purkinje and possibly Golgi Type-II cells) and fibroblasts. RCA 120 receptors are weakly expressed on astro-and oligodendroglia. Cell type-specific expression of RCA 120 receptors is constant throughout all ages studied (embryonic day 13 to postnatal day 9). At early embryonic ages the proportion of highly fluorescent neurons with large cell bodies is significantly increased.     

Evaluation of fluorescently labeled lectins for noninvasive localization of extracellular polymeric substances in Sphingomonas biofilms

Three strains of Sphingomonas were grown as biofilms and tested for binding of five fluorescently labeled lectins (Con A-type IV-TRITC or -Cy5, Pha-E-TRITC, PNA-TRITC, UEA 1-TRITC, and WGA-Texas red). Only ConA and WGA were significantly bound by the biofilms. Binding of the five lectins to artificial biofilms made of the commercially available Sphingomonas extracellular polysaccharides was similar to binding to living biofilms. Staining of the living and artificial biofilms by ConA might be explained as binding of the lectin to the terminal mannosyl and terminal glucosyl residues in the polysaccharides secreted by Sphingomonas as well as to the terminal mannosyl residue in glycosphingolipids. Staining of the biofilms by WGA could only be explained as binding to the Sphingomonas glycosphingolipid membrane, binding to the cell wall, or nonspecific binding. Glycoconjugation of ConA and WGA with the target sugars glucose and N-acetylglucosamine, respectively, was used as a method for evaluation of the specificity of the lectins towards Sphingomonas biofilms and Sphingomonas polysaccharides. Our results show that the binding of lectins to biofilms does not necessarily prove the presence of specific target sugars in the extracellular polymeric substances (EPS) in biofilms. The lectins may bind to non-EPS targets or adhere nonspecifically to components of the biofilm matrix.     

Evaluation of Fluorescently Labeled Lectins for Noninvasive Localization of Extracellular Polymeric Substances in Sphingomonas Biofilms

Three strains of Sphingomonas were grown as biofilms and tested for binding of five fluorescently labeled lectins (Con A-type IV-TRITC or -Cy5, Pha-E-TRITC, PNA-TRITC, UEA 1-TRITC, and WGA-Texas red). Only ConA and WGA were significantly bound by the biofilms. Binding of the five lectins to artificial biofilms made of the commercially available Sphingomonas extracellular polysaccharides was similar to binding to living biofilms. Staining of the living and artificial biofilms by ConA might be explained as binding of the lectin to the terminal mannosyl and terminal glucosyl residues in the polysaccharides secreted by Sphingomonas as well as to the terminal mannosyl residue in glycosphingolipids. Staining of the biofilms by WGA could only be explained as binding to the Sphingomonas glycosphingolipid membrane, binding to the cell wall, or nonspecific binding. Glycoconjugation of ConA and WGA with the target sugars glucose and N-acetylglucosamine, respectively, was used as a method for evaluation of the specificity of the lectins towards Sphingomonas biofilms and Sphingomonas polysaccharides. Our results show that the binding of lectins to biofilms does not necessarily prove the presence of specific target sugars in the extracellular polymeric substances (EPS) in biofilms. The lectins may bind to non-EPS targets or adhere nonspecifically to components of the biofilm matrix.     

DISTINCTIVE LECTIN LABELING OF THE FLAGELLAR APPARATUS OF TETRASELMIS (PRASINOPHYCEAE) AND DUNALIELLA (CHLOROPHYCEAE)1

Fluorescent labeling of the flagellar apparatus of Tetraselmis (Prasinophyceae) and Dunaliella (Polyblepharidaceae, Chlorophyceae) were successfully performed using fluorescein isothiocyanate–labeled lectins from Arachis hypogaea and Glycine maxima. These lectins specifically bound to the flagella and kinetosome of the cell but did not bind to the cell surface. Lectin binding on the flagellar apparatus remained constant under different culture media, temperatures, irradiances, cell division cycle, and culture aging. All the Tetraselmis and Dunaliella analyzed (five species, 20 clones) showed intense labeling of the flagellar apparatus. In contrast, no other species analyzed (46 clones of 25 species from four classes) showed binding to their flagellar apparatus. After the lectin treatment, many cells remained alive, and they were able to swim with the flagellar apparatus intensely labeled. The lectin binding indicates that the flagella and kinetosome of Tetraselmis are rich in Gal and GalNH₂ moieties and that the flagella of Dunaliella are rich in Gal and GalNAc moieties. Apparently, this feature seems to be specific to these species.     

Cell repair through cell fusion in the red algaGriffithsia pacifica

Summary When an intercalary shoot cell of the red algaGriffithsia pacifica is killed, the cell may be replaced through the wound-healing process of cell repair. During cell repair the cells on either side of the dead cell cut off new cells towards the dead cell. The superjacent cell produces a rhizoid; the subjacent cell produces an atypical shoot cell. The two new cells grow towards each other through the lumen of the dead cell. When they meet, they fuse; the resulting cell expands laterally to fill the cavity of the dead cell and is transformed into a typical intercalary shoot cell, morphologically and physiologically indistinguishable from the killed cell it replaces. The entire cell repair process takes 24–30 hours. Three aspects of cell repair suggest that intercellular communication occurs across the dead cell; these are a precocious division of the cell below the dead cell, a reversible change in the morphology and growth of the shoot cell which participates in repair, and a definite attraction between the two cells which fuse. Thus during cell repair we find evidence not only for cellular redifferentiation through cell fusion, but also for extracellular substances which change pathways of morphogenesis.     

Lectin-binding glycoconjugates in Paramecium primaurelia: changes with cellular age and starvation

 Lectins with different sugar specificities and binding to phagosome-lysosome systems as well as cell surface constituents were used to study glycoconjugate variation throughout culture and clonal life in Paramecium primaurelia, particularly during the transition period from logarithmic to stationary growth phase and in relation to clonal decline, respectively. These lectins include Griffonia simplicifolia agglutinin II (GS II), Ricinus communis agglutinin (RCA₁₂₀), Arachis hypogea agglutinin (PNA), succinyl concanavalin A (succinyl-con A), and Triticum vulgaris agglutinin (WGA). The labeling obtained varies both according to the lectin used and to the culture and clonal age of the cells. Negative results were obtained in logarithmic growth phase cells and in clonal young cells by using lectin GS II. Conversely, lectins RCA₁₂₀ and PNA bind to the cell surface, the oral region as well as cilia, and do not undergo modifications with culture or clonal age and after permeabilization. WGA binds to constituents of the cell surface, trichocyst tips, food vacuoles, the oral region, and cilia but the extent of labeling decreases as culture age increases; during clonal decline, cells show the same labeling pattern as starved cells. Finally, the lectin succinyl-con A shows a large amount of binding sites on the cell surface, on trichocyst tips, and in the oral region of logarithmic-phase cells, whereas the number of sites decreases in late stationary phase. The data obtained partly differ from those reported in the literature and the differences can be attributed to the culture conditions and species examined. Nevertheless, the assumption that a rearrangement of some glycoconjugates of membrane throughout culture and clonal life of Paramecium is confirmed. Accepted: 25 November 1996     

Glycoconjugates in normal human kidney. A histochemical study using 13 biotinylated lectins

The avidin-biotin-peroxidase complex technique was used with 13 lectins to study the glycoconjugates of normal human renal tissue. The evaluated lectins included Triticum vulgaris (WGA), Concanavalin ensiformis (ConA), Phaseolus vulgaris leukoagglutinin and erythroagglutinin (PHA-L and PHA-E), Lens culinaris (LCA), Pisum sativum (PSA), Dolichos biflorus (DBA), Glycine max (SBA), Arachis hypogaea (PNA), Sophora japonica (SJA), Bandeiraea simplicifolia I (BSL-I), Ulex europaeus I (UEA-I) and Ricinus communis I (RCA-I). Characteristic and reproducible staining patterns were observed. WGA and ConA stained all tubules; PHA-L, PHA-E, LCA, PSA stained predominantly proximal tubules; DBA, SBA, PNA, SJA and BSL-I stained predominantly distal portions of nephrons. In glomeruli, WGA and PHA-L stained predominantly visceral epithelial cells; ConA stained predominantly basement membranes and UEA-I stained exclusively endothelial cells. UEA-I also stained endothelial cells of other blood vessels and medullary collecting ducts. Sialidase treatment before staining caused marked changes of the binding patterns of several lectins including a focal loss of glomerular and tubular staining by WGA; an acquired staining of endothelium by PNA and SBA; and of glomeruli by PNA, SBA, PHA-E, LCA, PSA and RCA-I. The known saccharide specificities and binding patterns of the lectins employed in this study allowed some conclusions about the nature and the distribution of the sugar residues in the oligosaccharide chains of renal glycoconjugates. The technique used in this report may be applicable to other studies such as evaluation of normal renal maturation, classification of renal cysts and pathogenesis of nephrotic syndrome. The observations herein reported may serve as a reference for these studies.     

A Highlights from MBoC Selection: A central role for vimentin in regulating repair function during healing of the lens epithelium

Mock cataract surgery provides a unique ex vivo model for studying wound repair in a clinically relevant setting. Here wound healing involves a classical collective migration of the lens epithelium, directed at the leading edge by an innate mesenchymal subpopulation of vimentin-rich repair cells. We report that vimentin is essential to the function of repair cells as the directors of the wound-healing process. Vimentin and not actin filaments are the predominant cytoskeletal elements in the lamellipodial extensions of the repair cells at the wound edge. These vimentin filaments link to paxillin-containing focal adhesions at the lamellipodial tips. Microtubules are involved in the extension of vimentin filaments in repair cells, the elaboration of vimentin-rich protrusions, and wound closure. The requirement for vimentin in repair cell function is revealed by both small interfering RNA vimentin knockdown and exposure to the vimentin-targeted drug withaferin A. Perturbation of vimentin impairs repair cell function and wound closure. Coimmunoprecipitation analysis reveals for the first time that myosin IIB is associated with vimentin, linking vimentin function in cell migration to myosin II motor proteins. These studies reveal a critical role for vimentin in repair cell function in regulating the collective movement of the epithelium in response to wounding.     

179 Conjugation Process of a Freshwater Green Alga, Spirogyra Varians (Zygnematales, Charophyceae Examined by Fitc-Lectins)

The conjugation processes of filamentous freshwater green alga Spirogyra varians were examined by the use of FITC-lectins. Conjugation comprised of five steps: 1) array with adjacent filaments, 2) formation of conjugation protrusion (papilla), 3) fusion of the protrusions, 4) formation of conjugation tube, and 5) formation of zygotes. Three lectins, ConA, RCA and UEA, showed considerable labeling during the progression of conjugation. FITC-ConA labeled the surfaces of filaments throughout the whole conjugation processes. No labeling of FITC-RCA was detected on the surface of vegetative filaments. FITC-RCA labeling was observed at the conjugation protrusions only after the papilla formation. Strong labeling continued until formation of zygotes even in hollow area between the conjugation tube. The labeling decreased gradually over time and disappeared when zygotes were formed. FITC-UEA showed similar labeling pattern with FITC-RCA except that the labeling did not disappear even after zygote formation. Inhibition experiments using D-galactose, L-fucose and D-mannose, which are complementary carbohydrates for the above lectins, showed considerable decrease of conjugation (<50% vs. 83% in control). Hamagglutination experiment using crude extract of Spyrogyra varians revealed existence of lectins specific for the above carbohydrates. These results suggested that the lectin-carbohydrate recognition system might be involved in the conjugation of Spirogyra varians .     

A lectin cytochemical study of glycoconjugates in the human retina

Summary The binding to morphologically normal human retina of eleven biotin- or peroxidase-coupled lectins with different carbohydrate specificities was studied. Eight formalin-fixed and paraffin-embedded eyes were examined. Photoreceptor cells bound Lens culinaris (LCA), wheat germ (WGA), peanut (PNA) and Ricinus communis (RCAI) agglutinins, and concanavalin A (ConA). The outer segment region was labeled more strongly than the inner segment region, and PNA labeled only cones. All these lectins except PNA bound to both plexiform layers, and all but PNA and RCAI to the nuclear layers. Pretreatment with neuraminidase to remove sialic acid resulted in increased binding of RCAI and PNA, which now labeled both rods and cones, and in decreased binding of WGA. Bandeiraea simplicifolia (BSAI), Dolichos biflorus (DBA), soybean (SBA), Ulex europaeus (UEAI), and Lotus tetragonolobus (LTA) agglutinins, as well as pokeweed mitogen (PWM) reacted only with retinal vascular endothelial cells, which were also labeled with the other lectins. The results indicate that -mannose, -glucose, -galactose, N-acetyl-d-glucosamine and N-acetylneuraminic acid are present in glycoconjugates of human neuroretina.     

Lectin histochemistry of human placenta

Abstract. The human placenta was studied histochemically using 23 fluorescein-isothiocyanate-labeled lectins Distinct patterns of staining, as well as some differences between first-trimester and term placenta, were discerned. Eleven lectins (HPA, VVA, BPA, HAA, SBA, PNA, GSA-I, MPA, RCA-I, RCA-II, and UEA-I) did not react with the trophoblast. Two lectins (LCA and PEA) reacted with the trophoblast of first-trimester placenta but not with the trophoblast of third-trimester placenta. The remaining ten lectins (ConA, Suc.ConA, WGA, GSA-II, LAA, STA, DBA, LBA, PHA-E, and PHA-L) reacted with the trophoblast of both first- and third-trimester placenta, and two of these lectins (ConA and Suc.ConA) reacted preferentially with the syncytiotrophoblast. Five lectins (LAA, STA, DBA, GSA-II, and LBA) reacted with nuclei of the cytotrophoblast. The nuclei of some stromal and syncytiotrophoblastic cells were also reactive. Eighteen lectins reacted with the trophoblastic basement membrane, and all reacted with Hofbauer cells and the stroma of the villi. Latin binding was influenced by the mode of fixation and tissue processing. These data show that some lectins can be used to identify components of the placental villi (e.g., basement, membrane) to characterize differences between the first- and third-trimester trophoblast, and to distinguish the cytotrophoblast from the syncytiotrophoblast.     

Importance of N-linked sugar residues in the development of Auerbach's plexus in the rat colon: a lectin histochemical study

The lectin-binding patterns in Auerbach's plexus in the distal portions of the rat colon from 15- to 21-day-old foetuses, newborns, and adults were examined by light and electron microscopy using 16 different lectins (ConA, RCA-1, WGA, PNA, SBA, UEA-1, DBA, LCA, PHA-L, DSA, GS-1, VVA, MPA, BPA, MAA, and PSA). The binding of ConA was shown to increase after day 19 of gestation in parallel with differentiation of Auerbach's plexus, whereas the staining intensity for DSA and RCA-1 increased after day 17 of gestation in accordance with the appearance of the plexus. At the electron microscopical level, DSA binding sites were observed to be localized mainly in the plasma membrane, Golgi apparatus, and nuclear membrane of nerve cells. Positive sites were also observed in the axolemma and in the plasma membrane of nerve cell processes, Schwann cells, and the surrounding smooth muscle cells. PSA, PHA-L, LCA, and WGA showed constant staining during the development after day 15 of gestation. Other lectins, most of which are specific for O-glycosidic mucin-type sugar residues, were essentially negative throughout the developmental stages. Moreover, N-glycanase digestion significantly diminished the positive reactions. N-linked oligosaccharides may thus play important roles in the development and maturation of the Auerbach's plexus, and may be involved in the developmental defect of the plexus, e.g. as occurs in Hirschsprung's disease.     

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 12 h, rapidly reached a maximum at about 18 h and then declined to near zero at 48 h. About 28% of the nuclei were EdU labeled at 18 h reflecting the proportion of cells in S-phase of the cell cycle. During the ∼30 h 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.