Studies on the Distribution and Properties of a New Class of Cell Division-Promoting Substances From Higher Plant Species

Abstract

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

Lack of Cytokinin Activity of Quinolinol Sulfate

“…The findings of these axenic bioassays, in particular the moss bud test which is extremely sensitive to all purine-type cytokinins (20), strongly indicate that 8-quinolinol sulfate is not a true cytokinin and confirm the earlier suggestion by Rameshwar and Steponkus (16). The chlorophyll retention assay employed by Chua (4) suffers from a lack of specificity (11).…”

“…The assay employing moss protonema was carried out as described by Brandes and Kende (2) on the media of Wood et al (20). Spores of Funaria hygrometrica (L.) Sibth, also supplied by Dr. H. Kende, were grown in Petri plates for 26 days on the basal medium, and then bits of the protonematal mass were transferred by use of a sterile scalpel to plates containing the basal medium plus test substances for 5 to 6 days, at which time buds were counted.…”

“…Acme) was obtained from Dr. H. Kende, AEC Plant Research Laboratory at Michigan State University. Pieces of callus, approximately 1.0 x 0.5 cm, were placed on 50 ml of solidified media (13) in 125-ml Erlenmeyer flasks and grown under continuous light for 6 to 7 weeks, when final weights were taken.The assay employing moss protonema was carried out as described by Brandes and Kende (2) on the media of Wood et al (20). Spores of Funaria hygrometrica (L.) Sibth, also supplied by Dr. H. Kende, were grown in Petri plates for 26 days on the basal medium, and then bits of the protonematal mass were transferred by use of a sterile scalpel to plates containing the basal medium plus test substances for 5 to 6 days, at which time buds were counted.…”

Bioassay and Attributes of a Growth Factor Associated with Crown Gall Tumors

“…No characteristics other than tumor growth promotion can as yet be directly associated with the TGF. However, the absence of cytokinin-like activity in purified TGF preparations and the absence of a nicotinamide-like spectrum in the partly purified TGF indicates that it is not the unique cytokinin with a nicotinamide-like spectrum reported from normal and crown-gall tumor tissues by Wood et al (23).…”

“…The absence of TGF in extracts of several nongrowing healthy tissues, coupled with the discovery of TGF activity in growing tissues of healthy pinto beans, tobacco, and Bryophyllum, however, strongly suggests that TGF is a misnomer and in reality the active component is a normal plant growth factor whose synthesis is increased on infection with Agrobacterium sp. In view of the characteristic promotion of auxin and cytokinin synthesis associated with crown gall tumors (2, 10,22,23) and the growth characteristics of these tumors it would not be surprising to find that the synthesis of additional plant growth factors is activated by these infections and that one or more of these have yet to be described. Since many factors may promote tumor growth and the active material in these healthy tissue extracts has not been shown to be identical with TGF, the active material from the two sources can only tentatively be considered identical.…”