PROTEIN‐BOUND SULFHYDRYL GROUPS IN COLEUS WOUND MERISTEMS

ROBERTS, LORIN W. (U. Idaho, Moscow.) Protein-bound sulfhydryl groups in Coleus wound meristems. Amer. Jour. Bot. 47(2): 110-114. Illus. 1960.-A histochemical examination was conducted of the developmient of the Item wound-meristem of Coleus blumei Benth. with 2,2'-dihydroxy-6,6'-dinaphthyl disulfide (DDD) and 1(4-chloromereciriphenylazo) -naphthol-2 (Mercury Orange) to test for protein-bound sulfhydryl groups. Specificity for the histochemical reactions was indicated by complete blockinfgf of staining in sections pretreated with 0.001 M iodine in npropanol and 0.1 M aqueous solutions of iodoacetamide. Both histochemical methods yielded comparable results in protein-SH localization (zone of cell division and differentiating xylem elemments) and staining intensity. The zone of cell division did not exhibit staining at the time of initial cell division (3 days). However, the cytoplasm of the dividing cells of the wound-meristem demonstrated protein-SH 4-5 days following wounding. A high concentration of protein-SH was observed in the cytoplasm of the dividing cells 7. days after wounding. Redifferentiating parenchyma cells, destined to become wound-xylem elements, first exhibited wall staining at the time of protoplast contraction. The subsequent walled xylem stage exhibited strong staining in the reticulated striations of the secondary w alls. THE FREQUENT literature references to the possible role of sulfhydryl in cell division are too wellknown to reiterate in detail (Barron, 1951). Although determinations have been made on proteinbound SH during cell division, there has been little experimental evidence to demonstrate significant changes of this chemical group during division. Mazia (1954) reported evidence that protein-SH may have a structural function by the oxidative formation of the disulfide linkage in the mitotic spindle. However, he could find no significant difference in protein-SH staining between the cytoplasm of nondividing cells and cells at various stages of mitosis using the DDD histochemical procedure of Barrnett and Seligman (J952). Similarly, amperometrically determined protein-SH indicated no significant chanre in concentration during mitosis of microspores of Lilium longiflorum var. 'Croft' (Stern, 1956). Previous results from my laboratory, employing 2,2'-dihydroxy-6,6'dinaphthyl disulfide (DDD method of Barrnett and Seligman, 1952), 1(4-chloromercuriphenylazo) naphthol-2 (Mercury Orange method of Bennett, 1951), and 4-iodoacetamido-naphthol-1 (IAN method of Roberts, 1956), have demonstrated a high concentration of protein-SH in apical meristems, embryonic tissues, and the vascular cambium of higher plants (Roberts and Lucchese, 1955; Roberts, 1956). These results suggested that changes in protein-SH during cell division in plant tissues might be approached by investigating a stem wound-meristem. By employing this approach, changes in protein-SH could be determined prior 1Received for publication June 7, 1959. This investigation was supported by a research grant (G-5708) from the National Science Foundation. The writer wishes to express his appreciation for the services of Miss M. S. Dempsey, Department of Botany, University of Michigan, for her technical assistance. to, during, and following cell division. Followirng the act of wounding, one could predict the histological location of the dedifferentiating parenchyma cells that would form the zone of cell division. Also the time required for the initiation of cell division could be determined with reasonable accuracy. The Coleus stem wound-meristem was chosen because it has been extensively investigated (Surrey, 1955) and the plant is easily propagated. This paper is devoted to a brief description of the changes in protein-SH content of the Coleus stem tissues during the development of the wound-meristem. MATERIALS AND METHODS. A single clone of Coleus blumei Benth.2 was vegetatively propagated in the University of Idaho greenhouse by taking repeated stem cuttings from the original plant. Approximately 100 plants were employed in the experiments described in this report. Although a considerable number of these plants were sacrificed for specificity tests and for developing suitable histochemical staining procedures, stem wound-meristems from approximately 60 plants were sectioned. Slides prepared from each stem wound-meristem were stained by both the Mercury Orange and DDD methods, respectively. Supplementarv lighting was required during the winter months to facilitate growth. The plants were maintained under reasonably uniform environmental conditions throughout the experimental period. For a given experiment, plants that had approximately the same stem diameter, internode lencth, and leaf area were selected fronm the same age group. These criteria applied to the second internode and second leaf pair of the test plants. Although Erickson and Miche2The experimental plant was taxonomnically identified by Prof. W. H. Snyder, Department of Horticulture, University of Idaho.