Cytokinins bioassays

Most of the cytokinin antagonists were identified based on their biological effect in various cytokinin bioassays. From structure-activity relationship studies and inhibitor-like effects, it was hypothesized that these compounds act as competitive inhibitors at the receptor level [28,31,32]. However, direct proof that the molecular target of these compounds is the cytokinin receptor has been hindered by the dearth of knowledge of cytokinin receptors and signaling. After the cytokinin receptors have been identified in Arabidopsis and maize [38-40], the mode of action of the above described types of anticytokinins can be revised. Our recent results show that the molecular target of some of the potent pyrimidine-derived anticytokinins is not the cytokinin receptor, but rather cell-division machinery (L, Spichal, unpublished). 

J.H. Yopp, Southern Illinois University, Carbondale, became interested in the brassins project in its early stages (36) and he conducted cooperative brassins studies with both Mitchell and Mandava. Yopp et al. (37, 38) evaluated brassins responses in a number of auxin, gibberellin, and cytokinin bioassays and showed that brassins responded similarly to some of the known hormones in certain systems but not in others. These studies led to more cooperative investigation of brassinolide and other brassinosteroids (39, 40). 

Letham DS (1971) Regulators of cell division in plant tissues. XII. A cytokinin bioassay using excised radish cotyledons. Physiol Plant 25 391-396 Libbenga KR, Xorrey JG (1973) Hormone-induced endoreduplication prior to mitosis in cultured pea root cortex cells. Am J Bot 60 293-299 Lieberman M, Kunishi AX (1971) Abscisic acid and ethylene production. Plant Physiol 47, Suppl, p 22... 

Malik CP, Mehan M (1975 b) Interaction between cycocel and gibberellic-acid in pollen-tube elongation of Calotropis procera. Curr Sci 44 785-786 Manos GE (1961) The effects of growth substances on attached and detached root tips of Pisum sativum L. Physiol Plant 14 697-711 Manos PJ, Goldthwaite J (1976) An improved cytokinin bioassay using cultured soybean hypocotyl sections. Plant Physiol 57 894-897 Marinos NG (1960) Some responses of Avena coleoptiles to ethylene. J Exp Bot 11 227-235... 

The observations that cytokinins have activity in the tobacco bioassay detectable at concentrations as low as 10 H M, and that the promotion of such activity can depend on structural parameters... 

Figure 1. Cytokinin activities in the tobacco bioassay of 6-(3-methyl-2-butenyl-amino)purine (3), 6-isopentylaminopurine, and four (3-methyl-)7-alkylaminopura-zolo[4,3-d]pyrimidine (11-14)...

The accumulated assay results obtained with the 4-alkyl-2-methylpyrrolo[2,3-d]pyrimidines are of special interest. Can-pounds 25 and 26 exhibited significant cytokinin activity in all assays utilized. On the other hand, compound 27 was a potent antagonist in the tobacco bioassay but had little activity in the other two assay systems, while 29 acted as an anticytokinin in the tobacco bioassay and a cytokinin in the lettuce seed germination assay, but was without activity in the Amaranthus test system. 

Cytokinins Lycopersicon esculentum none reverse phase c18 bioassay (47)... 

Figure 1. Effects of CaCl2 on bioassays for auxin, gibberellin, and cytokinin. A effects of CaCl2 on elongation of oat coleoptile sections in the presence and absence of indoleactic acid B effects on elongation of lettuce hypocotyls in the presence and absence of gibberellic add and C effects on enlargement of Xanthium cotyledon pieces in the presence and absence of benzyladenine (13).

Certain substituted urea compounds, such as phenylurea or thidiazuron. Fig. (1), are very effective in the replacement of adenine-based cytokinins in promoting callus growth and other bioassays [27,28]. Molecular modeling revealed that the energetically optimal conformation of active urea derivatives have similar geometry as the isoprenoid side chain, so they can bind to the active sites of cytokinin metabolic enzymes and/or activate cytokinin receptors [29]. Thus, these compounds are likely to enhance their cytokinin effect by simultaneous activation of the receptor and inhibition of some of the cytokinin deactivating enzymes [11,30]. 

Specificity. Using the first member of the family, brassinolide (BR), an extensive survey of its effects in 17 bioassays, which varied in their responses to gibberellins, auxins and cytokinins, showed that BR did not behave exclusively as any one of those hormones. In some supposedly specific bioassays BR was as effective, or more so, as the hormone the assay was supposed to detect (9,10). This also applies to the rice lamina inclination assay (11), which is now frequently used. 

A tremendous upsurge in synthetic activity in this field is evident. A stereoselective synthesis of cis-zeatin (22), whose geometrical isomer was first isolated from Zea mays and shown to be a stimulant of cell division in plant tissue cultures, has been reported (Scheme 2). The Diels-Alder reaction of 1-chloro-l-nitro-socyclohexane (19) with isoprene gave the dihydro-l,2-oxazine hydrochloride (20) in moderate yield. Liberation of the base of (20) followed by zinc-acetic acid reduction gave the required amino-alcohol (21), which upon treatment with 6-chloropurine provided a separable mixture of cis-zeatin (22) and trans-zeatin. Standard tobacco callus bioassay for cytokinin activity showed that the natural trans-zeatin is at least 50 times more active than the synthetic cis-isomer (22). 

Brassinolide was tested on 17 bioassays for growth substance. The results led to claims that brassinolide possesses a broad spectrum of biological activity, including gibberellin-, auxin- and cytokinin-like activity (32,34). These claims must be treated with some caution however, since the claimed "specificity of some of the bioassays selected is questionable. At present three bioassay techniques (35,36,37) are used routinely for the detection of brassinolide activity. All three assays are sensitive to auxin, which is a prerequisite for the detection of brassinolide-like compounds. This is not to say that brassinolide has auxin-like activity, but rather there seems to be an interaction of cooperative action between auxin and BR. 

The cytokinins are extremely potent as little as 5 X 10 M 6-(4-hydroxy-3-methyl-frans-2-butenylamino) purine can be detected by some bioassay systems. They have a wide variety of actions in plants, including promotion of cell division, delay of senescence, and resistance to adverse conditions. They affect plant metabolism, organelle development, and fruit and flower formation. Unfortunately, their basic mechanisms of action are not known. 

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