The Time Course of Xylem Differentiation and Its Relation to Deoxyribonucleic Acid Synthesis in Cultured Coleus Stem Segments

The relationship between DNA synthesis and wound xylem differentiation was investigated in cultured stem segments of Coleus blumei. The addition of 50 micrograms of indoleacetic acid per liter to the culture medium resulted in a 400 to 500% increase in the number ofwound vessel members formed in 7 days. However, the time course of wound vessel member formation was similar in segments cultured in the presence and absence of auxin. In either case, no wound vessel members appeared before the 3rd day of culture, while the majority of wound vessel members appeared on the 4th and 5th days of culture. 3H-Thymidine incorporation into DNA was used to measure changes in the DNA synthetic activity of the tissues during the culture period. Comparatively little 3H-thymidine incorporation occurred during the 1st day of culture. Maximum 3H-thymidine incorporation was observed on the 2nd day of culture, 2 days before the peak period of xylem differentiation. The rate of incorporation of 3H-thymidine into DNA decreased with increasing time in culture after the 2nd day. Auxin at 50 micrograms per liter had no effect on the time course of 'H-thymidine incorporation, although somewhat more 3Hthymidine was incorporated into DNA throughout the culture period in the presence of auxin. The magnitude of this effect was small when compared to the effect of auxin on xylem differentiation. The antimetabolite 5-fluorodeoxyuridine was shown to block DNA synthesis in the cultured stem segments. When the tissues were isolated on media containing 10-. M 5-fluorodeoxyuridine, wound vessel member differentiation was inhibited by approximately 80%, in both the presence and absence of auxin. Thymidine at 104 M completely overcame the 5-fluorodeoxyuridine inhibition of wound vessel member formation. 5-Fluorodeoxyuridine was effective in blocking xylogenesis only when this substance was supplied to the tissues during the early part of the culture period. 5-Fluorodeoxyuridine had no effect on xylem differentiation when it was applied after the 3rd day of culture. Although auxin has been shown repeatedly to act as a limiting factor for xylem differentiation (7, 12-14, 24, 25), we know very little about the nature of this regulation. Ultimately one would hope to elucidate the biochemical mechanism(s) by which auxin Cambridge, Massachusetts 02138 controls xylogenesis. To facilitate this achievement, it would be useful first to discover where auxin acts during xylem element formation. Is auxin directly involved in the initiation of xylem differentiation, or does it limit the rate of steps which occur after a cell is committed to differentiation, such as secondary wall deposition or lignification? It is well established that the composition of the secondary wall of tracheary elements in woody species is determined, in part, by the concentration of auxin in the tissues (3, 16). However, in this case auxin appears to regulate the proportions of cellulose, hemicellulose, and lignin in the wall, rather than to determine the rate at which these substances are deposited. The simple study of the time course of xylem differentiation, as influenced by auxin, might be expected to tell us something about the role of auxin in this process. If auxin limited xylogenesis by controlling the rate of secondary wall deposition, one might expect auxin to accelerate the rate at which xylem elements appeared, but to have relatively little effect upon the total number of elements that were formed in a given experimental system. Another process, apparently regulated by auxin, which has been shown to be an integral part of the ontogeny of xylem elements, is DNA synthesis. DNA synthesis appears to be involved in xylogenesis in at least two different ways. First, DNA synthesis would be expected to accompany the mitotic activity which preceeds and is necessary for the initiation of xylem differentiation (6, 8, 14). Secondly, in some species an increase in nuclear DNA content accompanies the maturation of xylem elements. List (15) observed that the nuclei of differentiating metaxylem elements in the roots of Zea frequently attained amounts of DNA up to 32 times the normal diploid level. Although there is little direct evidence for or against the hypothesis that auxin regulates xylogenesis by controlling DNA synthesis, a number of studies have shown that cell proliferation can be stimulated in intact or wounded plants by auxin application (1, 9, 11, 17, 20-22). Also, the work of Skoog and co-workers (4, 18) suggests that auxin may be directly involved in the regulation ofDNA synthesis. They found that the treatment of tobacco pith tissue with auxin alone brought about DNA synthesis in a significant proportion of the cells, although relatively few of these cells went on to divide. As is well known, both auxin and cytokinin were required for the complete division cycle in this tissue. In the present investigation, the time course of xylem differentiation and DNA synthetic activity was determined in cultured Coleus stem segments, and the effect of auxin upon these processes was examined. Additional evidence is presented that DNA synthesis is necessary for xylogenesis, but the data do not support the idea that auxin regulates xylem differentiation through its effect upon DNA synthesis, or through possible effects upon the rate of secondary wall deposition.