Naturally occurring isomers of the dehydrodiconiferyl glucosides have been isolated from Vinca rosea crown gall tumors and have been tested for cell division promoting activities in the tobacco pith and leaf assay systems. The enantiomeric isomers A and B are active, although they are required at concentrations up to 2 orders of magnitude higher than zeatin riboside to promote comparable growth. We estimate that the active dehydrodiconiferyl glucosides are present in rapidly growing tissues (tumor tissue, habituated tissue, cultured nontransformed tissue) in micromolar concentrations. In quiescent tobacco pith tissue, the levels of these compounds are reduced by a factor of 100. These results suggest that cytokinin may exert control ofcell division through the accumulation of molecules (the dehydrodiconiferyl glucosides) that are apparent cell wall components.Cytokinins are naturally occurring plant growth regulators that play a central role in the control of plant cell division (1, 2). In addition, these N6-substituted adenine derivatives are involved in the control of a variety of other phenotypese.g., initiation of shoot organogenesis and inhibition of leaf senescence (3,4). The molecular and biochemical bases of the cytokinin-mediated control of these processes remain unknown. Wood and colleagues (5-7) provided evidence for cell division factors isolated from Vinca rosea crown gall tumors that could substitute for cytokinins in the tobacco pith assay. We have now shown (8) that the original fraction contained, at least in part, two dehydrodiconiferyl glucosides (DCGs). Here we further characterize the DCGs D, E, F, and G and demonstrate cell division promoting activity for isomers A, B, and E as ascertained by both tobacco pith and leaf bioassay. (9) supplemented with 1.0 uM naphthalene acetic acid (NAA) and zeatin riboside (ZR) or the DCG isomers as indicated. In some experiments, the pith tissues were placed in plastic plates (60 x 15 mm) containing 4.0 ml of medium. The ZR and DCGs were filter-sterilized and added aseptically to cooling autoclaved medium. The tissues were grown at 25TC under cool white fluorescent lights with a light/dark cycle of 16:8 hr. They were observed regularly under a binocular dissecting microscope and harvested after 3 weeks. For leaf assays, 9-mm2 fiagments (1-2 mg) were excised from 12-to 15-cm-long leaves, taking care to remove major veins. The explants were then placed in plastic Petri plates (35 x 10 mm) containing 2.0 ml of test medium. Tissues were photographed and harvested after 3 weeks. For organogenesis assays, leaf explants (9 mm2) were placed into plastic Petri dishes (60 x 15 mm) containing 5 ml of LS medium lacking NAA but supplemented with ZR or DCG. These were incubated at 25°C with a light/dark cycle of 16:8 hr and monitored regularly. Final scoring was tabulated after 6 weeks.For histological examination, tissues were fixed in 3.5% glutaraldehyde in 0.05 M phosphate buffer (pH 6.8) and embedded in glycol methacrylate (JB-4, Polyscience, Warrington, P...

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The ability of two compounds, a cytokinin and an auxin, to stimulate tobacco cell growth and differentiation has been known for >30 years, but the molecular mechanism of this activation is still unknown. Previous reports of factors endogenous in crown gall tumors of Vinca rosea that could replace the cytokinin requirement in tobacco cell culture has motivated an investigation of these tissues. The optimization of a reverse-phase isolation scheme has led to the purification of sufficient material to allow for the identification of six different related compounds. The structures of two of these compounds have been assigned as a set of epimeric dehydrodiconiferyl alcohol P-D-glycosides. The structure of these compounds suggests that they would most likely be derived from the plant cell wall.The induction of crown gall tumors in dicotyledonous plants by Agrobacterium tumefaciens involves the transfer and incorporation of oncogenic DNA into the host cell (1, 2). The resulting biochemical changes that allow these cells to grow continuously on defined medium without exogenous phytohormones have been studied extensively (3,4), and recently genes transferred to the host have been found that code for enzymes involved in the biosynthetic production of cytokinins (5) and auxins (6). The transformed cell continuously produces these hormones and grows autonomously at a state of differentiation specified by the bacterial strain (7). While it is well established that the endogenous hormones can account for the tissue growth (8, 9), the molecular basis ofthis process is not yet known. Previous reports (10-12) of other growth factors present in Vinca rosea crown gall tumors have stimulated this investigation, which has resulted in the isolation and characterization of compounds capable of replacing the cytokinin requirement for tobacco cell growth in culture. MATERIALS AND METHODSTissue Cultures. Axenic cultures of V. rosea crown gall tumors were grown on White's basic medium (13) containing 1% Bacto-Agar (Difco). Six pieces of tumor tissue (=0.5 g each) were each placed in a culture dish (25 x 100 mm). Tumors were grown under constant illumination. After 4 weeks, at which time the tissue was at logarithmic phase growth, a small portion was used for subculture and the remainder was harvested for isolation.Bioassay. Stems of Nicotiana tabacum cv. Havana 425 were surface-sterilized by washing with Ivory soap, washed three times each with 7% bleach and 70% EtOH/H20 (1 min each), and three times with sterile distilled H20. Pith was isolated from mature internode sections, cut into -10-mg explants, and placed in shell vials (90 x 25 mm) containing 5 ml of agar-solidified LS medium (14) A 1-kg batch of harvested tumor cells was extracted twice with equivalent amounts (wt/wt) of 50% MeOH/H20. The filtrate (2-3 liters) was concentrated in vacuo at 35°C to a thick syrup. The crude extract was partitioned in CH2Cl2/ MeOH/H20 (7:13:8, vol/vol), the organic layer was discarded, and the aqueous layer was filtered through a bed of cel...

and Summary.-One member of a new class of cell division-promoting substances, which are nicotinamide derivatives, has been found to be present in dividing cells of tobacco and cactus. These plants are taxonomically far removed from one another and from Vinca rosea L., the plant species from which the new substances were first isolated. Because of their apparent wide distribution among dicotyledonous plant species, the question is raised as to whether the nicotinamide derivatives rather than the purine cytokinins may not, in fact, be the naturally occurring cell division factors that are directly involved in promoting cytokinesis in higher plant species. Unequivocal evidence is presented to show that the nicotinamide derivatives do not owe their biological activity to contamination by 6-substituted purine cytokinins.Two members of a new class of cell division-promoting substances have been isolated and partially characterized chemically from crown-gall tumor cells of Vinca rosea L.8 These substances, which are newly synthesized by the tumor cells as a result of their transformation, have been found to play a central role in the development of a capacity for autonomous growth of such cell types.2 They are responsible for keeping the tumor cells dividing persistently. Normal cells of the type from which the tumor cells were derived possess, on the other hand, an absolute exogenous requirement for either a 6-substituted purine cytokinin such as kinetin (6-furfurylaminopurine) or a naturally occurring cell division factor of the type discussed here, if cytokinesis is to occur in an otherwise suitable culture medium. It has been found,9 however, that normal cells that are stimulated to divide with kinetin synthesize a compound which is identical in its physical, chemical, and biological properties to one of the two cell division-promoting factors synthesized by the tumor cells in the absence of an exogenous source of kinetin or other 6-substituted purine derivative. This finding has led to the suggestion that kinetin may not itself be directly involved in promoting cell division, but rather that it acts indirectly to induce the synthesis by normal cells of a substance(s) that is directly involved in that process.The biologically active substances synthesized by tumor cells as a result of their transformation and by normal cells grown in the presence of kinetin contain nicotinamide, a glucose sugar moiety, a straight-chain fatty acid(s), sulfur in the form of sulfate or sulfornate, and one or more methyl groups. These are, therefore, types of compounds that are very different from the 6-substituted purine derivatives that have been known for more than a decade to promote division in cells of higher plant species.It is the purpose of the present paper to present evidence that the biologically 349