Evidence for the natural occurrence of zeatin and derivatives: compounds from maize which promote cell division.

Both purine (1, 10) and urea (3) derivatives have been shown by various bioassays to be active cytokinins, the latter leading Bruce and Zwar (3) to hypothesize -NH--CO-NH-R as the active portion of the cytokinin molecule. Currently, cytokinins which have been extracted from plant materials, particularly from transfer RNA, are all N6-substituted purines (7,14). Fox and Chen, after demonstrating that benzyladenine, an N6-substituted purine, is incorporated into transfer RNA, hypothesized that cytokinins structurally complete certain inactive transfer RNA molecules, allowing them to function normally (5). While there is disagreement about this mechanism (8), the importance of purines is undisputed.Chua (4), using the chlorophyll retention assay, has recently identified 8-quinolinol sulfate (Fig. 1), an antimicrobial agent, as a cytokinin with maximal activity at 0.1 to 1 mm. This chemical differs markedly in structure from the usual purine nucleus, and obviously could not function through incorporation into tRNA. Chua suggests, somewhat unclearly, that the controlling activity of 8-quinolinol sulfate may be as a chelator, interfering in specific protein-metal cofactor relationships. In contrast, Rameshwar and Steponkus (16) report that 8-quinolinol sulfate cannot replace kinetin in stimulating the growth of soybean callus.Since the establishment of 8-quinolinol sulfate as a true cytokinin would throw considerable doubt on current ideas of the mechanism of cytokinin action, a more thorough evaluation of this compound, including possible interactions with known cytokinins, is desirable. In view of the fact that the conflicting results mentioned previously are based on only one bioassay performed in each laboratory, we decided to use several assays, each of which is highly specific (11, 18), and conducted in axenic conditions: the growth of tobacco pith (15) and soybean callus (13), and the production of buds by moss protonema (2). METHODS AND MATERIALSThe tobacco callus bioassay was carried out on the media of Murashige and Skoog (15), with the addition of 2 mg IAA/ liter and 1.6% agar. Pith was excised, by using a cork borer, from randomized segments of ornamental tobacco stem (Nicotiana tabacum L. cv. Assimis, obtained from Pennsylvania State University), cut into 0.5 x 0.3 cm pieces and 1 piece placed on approximately 35 ml of medium in a Petri dish. Final weights were taken after 4 to 5 weeks of growth in the dark.Soybean callus (Glycine max (L.) Merr., var. 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 ...

supporting

confidence: 75%

Lack of Cytokinin Activity of Quinolinol Sulfate

Kraft

Ellis

1971

“…Aliquots (1-5 ,tl) were then withdrawn for injection onto the column. Miller (20,21), who also recognized the possibility of degradation arising from the production of heat during elution steps (20). To overcome these problems in our study, special techniques were used to prevent the accumulation of heat during elution.…”

Section: Methodsmentioning

confidence: 99%

N⁶-(Δ²-Isopentenyl)adenosine: Its Occurrence as a Free Nucleoside in an Autonomous Strain of Tobacco Tissue

Dyson

Hall

1972

Cytokinins from both the free nucleoside pool and the transfer RNA have been isolated and identified in a habituated strain of tobacco pith callus (Nicotiana tabacum [L] var. Wisconsin 38). The transfer RNA of this strain contains both N0-(A2-isopentenyl) adenosine and N'-(4-hydroxy-3-methylbut-2-cis-enyl) adenosine. The trans-hydroxylated derivative is absent from the transfer RNA of this dark-grown tissue. N'-(A'-Isopentenyl)- Hall [14]). This hydroxylated derivative appears to be restricted to the tRNA of higher plants. In addition, the 2-methylthio derivatives of both the parent compound (z.2-iA) and the hydroxylated derivative have been isolated from the tRNA of wheat germ (6).The trans-isomer of ribosylzeatin (trans-RZ) and/or its free base and nucleotide have been found as free compounds in chicory roots (5) (8) showed that the A2-iA of the tRNA of this tissue is synthesized by attachment of a A2-isopentenyl side chain to the preformed tRNA. The requirement for the exogenous cytokinin does not seem to relate directly to this biosynthetic mechanism. In search, therefore, of leads that bear on the mechanism of action of cytokinins we decided to compare the A2-iA content of an autonomous line derived from the Wisconsin 38 strain of tobacco pith callus tissue (11) that grows well in the absence of exogenously added cytokinins. We have found that the tRNA of this strain also contains A2-iA and cis-RZ in its tRNA, but in contrast to the cytokinin-requiring strain the autonomous tissue contains a significant level of A2-iA in the free state. MATERIALS AND METHODSTissue Culture Strains. Normal tobacco pith tissue has an absolute requirement of added cytokinins for growth (11). Occasionally this normal tissue spontaneously loses its cytokinin requirement. Such autonomous strains of tissue presumably meet their cytokinin requirement through an alteration in some metabolic process, although the mechanism(s) of this transformation is not known.The normal cytokinin dependent tobacco pith tissue (Nicotiana tabacum [L] var. Wisconsin 38) and the derived autonomous strain used in this study were gifts from J. E. Fox of the University of Kansas. The normal pith tissue was grown on a previously described (11) basal medium containing naphthaleneacetic acid (1 mg per liter) and 6-furfurylaminopurine (kinetin) (1 mg per liter). The autonomous strain was grown on the same basal medium without the growth factors. Tissues were harvested after 4 to 5 weeks growth in the dark.Bioassay. Aliquots of column fractions to be bioassayed for cytokinin activity were added to 25-ml flasks and dried in vacuo over P20,. A basal medium (11), complete except for cytokinin, was added (10 ml per flask), and the flasks were autoclaved at 18 lb, 121 C for 5 min. A cytokinin requiring strain of soybean cotyledon tissue (Glycine max [L.] Merril, var. Acme) was planted (0.5 mg pieces, three per flask), and after 30 days incubation in the dark at room temperature, the increase in fresh weight was compared to controls grown in the presence (...

“…An active component in the extracts has been identified as trans-ribosylzeatin, trans-RZ2 (Fig. 1), in the case of immature fruit (16,17,20) and chicory root (2), and as its dihydro derivative in the case of Lupin (14) (see review by Kende (12) for a more detailed account). Recently Dyson and Hall (5) reported the presence of i6Ado in an extract of an autonomous strain of tobacco tissue.…”

mentioning

confidence: 99%

“…We chose systems known to contain free trans-RZ, a mycorrhizal fungus, Rhizopogon roseolus (22) and corn kernels (16,20 …”

mentioning

confidence: 99%

See 1 more Smart Citation

trans-Ribosylzeatin

Miura¹,

Hall²

1973

When [8-14C]-N-(A'-isopentenyl) adenosine is incubated with the endosperm of corn (2 weeks after pollination), it is converted to ['4C]-N6-(4-hydroxy-3-methylbut-2-trans-enyl) adenosine, trans-ribosylzeatin. This biosynthetic step, N6-(A2-isopentenyl)adenosine to ribosylzeatin, also occurs in the mycorrhizal fungus, Rhizopogon roseolus.Cytokinin activity has been detected in extracts of various plants and plant organs. An active component in the extracts has been identified as trans-ribosylzeatin, trans-RZ2 (Fig. 1), in the case of immature fruit (16,17,20) and chicory root (2), and as its dihydro derivative in the case of Lupin (14) (see review by Kende (12) for a more detailed account). Recently Dyson and Hall (5) reported the presence of i6Ado in an extract of an autonomous strain of tobacco tissue. Of these naturally occurring cytokinins, trans-RZ exhibits the greatest activity in the tobacco callus bioassay system (9).As part of a study of the metabolism of cytokinins, we have investigated the biosynthesis of trans-RZ, and in this paper we describe its synthesis from the precursor, i6Ado. We chose systems known to contain free trans-RZ, a mycorrhizal fungus, Rhizopogon roseolus (22) and corn kernels (16,20