13.4—
Action of Cytokinins

13.4.1—
Introduction

The term cytokinin has been accepted practically universally as a generic name for substances which promote cell division and exert other growth regulatory functions in the same manner as kinetin. The synthesis and testing of compounds for cytokinin activity began with the discovery of kinetin (6-furfurylaminopurine). Today there are probably at least one hundred known synthetic and native cytokinins (Fig. 13.4).

Structural requirements for a high order of cytokinin activity generally include an adenine molecule with the purine ring intact and with an N⁶ substituent of moderate size. One exception to the requirement for a modified purine exists, notably diphenylurea and its derivatives. Certain other substances which lack a true purine ring, such as 8-azakinetin, 6-benzylamino-8-azapurine and 6-(3-methyl-2-butenylamino)-8-azapurine are active, but each of them is less than 10% as active as its corresponding purine derivative. Substitution of either O or S for N in the N⁶ position of adenine in each case results in a more than 90% loss in growth promoting activity in the tobacco bio-assay.

Cytokinins play a role in practically all phases of plant development from cell division to the formation of flowers and fruits. They affect metabolism including the activities of enzymes and the biosynthesis of growth factors. They also influence the biogenesis of organelles and the flow of assimilates and nutrients within the plant. Cytokinins defer senescence and may protect the plant against adverse environments, such as water stress. These many diverse effects presumably stem from some primary anabolic function of the cytokinins that remains to be elucidated.

Figure 13.4
Structure of some common
native and synthetic cytokinins.

Cytokinins have been found in certain transfer-RNA (t-RNA) molecules from a large number of organisms including bacteria, yeast, plants and animals (see Cherry & Anderson, 1971). In sequence analysis the native cytokinin, isopentenyl adenine, always occurs adjacent (3' side) to the anticodon (Fig. 13.5). Furthermore, the location of the isopentenyl group is required for the

Figure 13.5
Diagram of the cloverleaf shape of seryl-transfer RNA.
The IPA residue is located adjacent to the anticodon.

specific tRNA to function efficiently in protein synthesis. Results involving bacteriophage infection of bacteria indicate that cytokinin in tRNA may have an important regulatory function in protein synthesis. In soybean seedlings (Anderson & Cherry, 1969) the application of a cytokinin (6-benzyladenine) results in increased levels of two species of leucyl-tRNAs. Although the relationship of cytokinins to tRNA may not be directly associated with the primary action of cytokinins on cellular activity, the alteration of specific tRNAs could control protein synthesis. Such a regulatory mechanism would be manifested in the control of growth and morphogenesis as the ultimate expression of the hormone.

13.4.2—
Actions of Cytokinins on Enzymes

Cytokinins have been shown to influence the activities of a number of specific enzymes. A most thoroughly studied case is the promotion by cytokinins of tyramine methylpherase activity in roots of germinating barley embryos. This appears to involve de novo synthesis although this conclusion is based solely on

the results of inhibitor experiments (Mann et al., 1963; Steinhart et al., 1964). Activities of four other enzymes tested were unaffected by kinetin treatment. The selective action on methylpherase is most likely related to the developmental stage of the seedling material and not to a specific affinity of cytokinin for the enzyme. Substances other than cytokinins (auxins and lysine) which also stimulated the appearance of tyramine methylpherase activity either were less effective or acted only during the early stages of germination. An analogous effect of kinetin in promoting synthesis of alkaloids in the roots of lupin has also been reported (see Skoog & Armstrong, 1970).

The induction of a -amylase activity in half-seeds without embryos, a well known effect of gibberellins in barley, is achieved in wheat also by treatment with cytokinins (Boothby & Wright, 1962). However, the effect of cytokinins on wheat is much less marked than that of gibberellin in the barley. In barley seeds, cytokinins cannot replace gibberellins, but do counteract an inhibition of gibberellin action by abscisic acid (Khan, 1969). The formation of amylase activity in excised hypocotyls of pea is stimulated by cytokinins but not by gibberellins or auxins (Chum, 1967). In germinating squash seeds cytokinins, but not gibberellins, at least partly substitute for the embryo in promoting the formation of isocitrate lyase and proteinase activity in the cotyledons.

The synthesis of deoxyisoflavones in soybean callus tissue is promoted by both cytokinins and auxins (Miller, 1969). The ease with which the compounds can be detected and the rapidity of the response suggests that this system might be useful as a rapid bioassay for cytokinins.

The rates of synthesis of ribulose bisphosphate carboxylase and NADP-dependent glyceraldehyde-phosphate dehydrogenase, studied in rice seedlings, appear to be under cytokinin control (Feierabend, 1969). Kinetin did not affect these enzymes until 96 hours after the beginning of germination. By contrast the activity of the cytoplasmic enzyme 6-phosphogluconic acid dehydrogenase was promoted by kinetin only during early stages of germination. It was concluded that cytokinins probably are not directly involved in gene derepression although the level of cytokinin may determine the extent to which the appropriate genes are expressed.

13.4.3—
Nucleic Acid Synthesis

Early studies with tobacco pith tissue showed that a proportion of the tetraploid cells present would be stimulated by kinetin treatments to enter mitosis and undergo cytokinesis without incorporation of thymidine. Thus the effect of kinetin on cell division was not always associated with DNA synthesis. In Lemna cultures 6-benzyl-aminopurine reverses the effect of abscisic acid in inhibiting growth and nucleic acid synthesis (van Overbeek et al., 1967). DNA synthesis in response to these growth substances appears to be more rapid than RNA synthesis. In axillary buds of tobacco which are normally inhibited by

the apex, treatment with 6-benzylaminopurine initiates DNA synthesis and growth (see Skoog & Armstrong, 1970).

Some inhibitors such as chloromycetin and 6-azauracil uncouple the two processes, preventing growth but not the cytokinin-induced DNA synthesis. Nevertheless, in view of the early work, it seems likely that cytokinin effects on DNA synthesis are indirect. In a detailed study of tuberization, Palmer and Smith (1969) have shown that, in excised stolons of potato, cytokinins inhibit stolon elongation and induce tuber formation. Other factors which inhibit elongation may also increase the inductive response in response to cytokinins, but they are not themselves inductive factors. Inhibitor studies suggest that cytokinin action is associated with protein synthesis or activation but not with DNA or messenger-RNA synthesis. Effects of cytokinin on RNA and protein synthesis and isolated cell organelles have been reported, but have not yet been analyzed in depth (Skoog & Armstrong, 1970). Nuclei preparations separated from coconut milk and incubated in the presence of kinetin showed an increased incorporation of labelled precursors into RNA and protein (Datta & Sen, 1965).

The effect of kinetin on RNA synthesis in an in vitro system containing purified chromatin and E. coli RNA polymerase has been studied by Matthysse and Phillips (1969). In the presence of a particular protein fraction, kinetin stimulated RNA synthesis. A similar effect of auxin was also mediated by a protein fraction. The effects of cytokinin in this system have not yet been studied in great detail. Fellenberg (1969) has observed that in vitro bindnig of kinetin to chromatin of pea epicotyls caused a slight decrease in the melting point of the nucleoprotein complex.

13.4.4—
Role of Cytokinins on Transfer RNA

One suggestion has been that the endogenous cytokinins somehow exert their activity by being incorporated into transfer-RNA (Skoog & Armstrong, 1969). For example, the synthetic cytokinin, benzyladenine, was reported to be incorporated into the tRNA of callus tissue (Fox & Chen, 1967). Richmond et al. (1970) specifically looked for incorporation of labelled benzyladenine into the RNA of senescing leaves, but found no evidence for incorporation. On the other hand, under similar conditions kinetin was incorporated into the RNA. Recently Walker et al. (1974) have shown that double-labelled benzyladenine is incorporated into tRNA at a frequency of one per 10⁴ molecules.

Chen and Hall (1969) have shown that cytokinin-dependent tobacco callus tissue contains the enzyme system which attaches the D² -isopentenyl group to the appropriate adenine residue in tRNA. In the case of tobacco callus tissue grown in the presence of radioactive benzyladenine, the tRNA still contained its complement of isopentenyl adenine derivatives even though the benzyladenine was incorporated into the tRNA. In general, various purine analogues can be incorporated into RNA in a rather non-specific way.

It is also quite possible that incorporation occurs into RNA fractions other

than tRNA, and unless the tRNA is rigorously characterized one cannot exclude the possibility of error arising by contamination with other RNA fractions. To establish whether the incorporation of N⁶ -substituted adenosine derivatives into tRNA has a biological significance it would be necessary to fractionate the tRNA and identify the incorporated material at a specific location in the tRNA sequence.

13.4.5—
Model for Cytokinin Action

Cytokinins are found in transfer-RNAs of animals, microorganisms and plants (see Cherry, 1973). Furthermore, in those cases where the nucleotide sequence is known the native cytokinin, IPA (isopentenyladenosine), or a modified form, is found adjacent to the anticodon. At the moment it appears that all IPA containing species of tRNA recognize codon triplets which begin with U (Skoog & Armstrong 1970). Thus, the anticodon loop of a cytokinin-containing RNA could be one of at least sixteen codon sequences of the following type: A— —IPA. By eliminating the nonsense terminator and the phenylalanine codons, it seems that IPA-containing tRNAs would be confined to less than ten codons (see Table 13.1).

Table 13.1. Codons beginning with U.

It may be that IPA adjacent to the anticodon of these tRNA species plays some role in messenger-RNA-tRNA-ribosome recognition, and that these tRNA species are required for the translation of some messenger-RNAs into specific proteins. Based on these assumptions it is possible to speculate on the mechanism of action of cytokinins in plants. From the observation that cytokinins appear not to be incorporated into tRNA, as precursor units, but rather the isopentenyl group comes from mevalonic acid (Chen & Hall, 1969), it seems likely that cytokinins have no effect on the synthesis of tRNA containing IPA. Furthermore, a number of chemicals, including various isomers of IPA, kinetin, 6-benzyladenine and even diphenylurea, have cytokinin biological activities, and it appears that these cytokinins do not participate in tRNA synthesis. If this is true, then what is the mechanism of cytokinin action?

A model presented in Fig. 13.6 is based on the speculation that specific

Figure 13.6
A model of cytokinin action. Based on the fact that tRNA specific nucleases
are known, it is suggested that free cytokinins protect cytokinin-containing
tRNAs by inhibiting nuclease activity, specific as well as general.

nucleases break the primary structure of the IPA containing tRNA (Cherry & Anderson, 1971). The IPA would provide a unique attachment for the enzyme to bind to and subsequently to break the phosphodiester bonds. The action of this enzyme would destroy the function of the tRNA and yield free cytokinin. Cytokinins including 6-benzyladenine and diphenylurea added exogenously to plants or tissue cultures would defer senescence by essentially protecting the cytokinin-containing tRNA species. This protection would be mediated by the exogenously added cytokinin binding to the nuclease and competitively inhibiting its action. Alternatively, the cytokinin could bind to other agents which in turn prevent an increase in nuclease activity which would destroy the cytokinin-containing tRNA. Therefore, tissue treated with cytokinin would retain its IPA-containing tRNA species and continue to synthesize essential proteins as long as sufficient free cytokinin was present within the tissue.

Preliminary unpublished work of Locy (1974) provides suggestive evidence of a tRNA-specific nuclease. We have found that chloroplasts of soybeans contain a nuclease which specifically degrades one of the three tyrosyl-tRNAs. At the moment we do not have any evidence to suggest that free cytokinin controls the activity of this tRNA nuclease. In regards to this model it is to be emphasized that it is hypothetical and is presented as a working base upon which to investigate further the mechanism of action of cytokinins at the molecular level.

13.4.6—
Summary

Cytokinins have been shown to increase the activities of a number of enzymes. These increases in activity do not appear to be a direct effect of the cytokinin

but rather to be an indirect effect of some kind. Cytokinins increase the amount of DNA produced in a number of tissues. Furthermore, cytokinins increase the rate of RNA synthesis in ageing tissue. However, a number of studies have pointed out that the cytokinin effect on nucleic acid synthesis most likely is not due to a direct effect on the derepression of the DNA within the genome.

Over the past few years there has been a great deal of excitement generated relating to the fact that the native cytokinin is found in some transfer-RNA isoaccepting species. For a few years data was presented which showed that cytokinins, particularly the synthetic cytokinin, 6-benzyladenine, was incorported directly into transfer-RNA. That excitement, however, appears to be shortlived since other studies have shown that the incorporation of the labelled synthetic cytokinins is not into tRNA species but into heavier forms of RNA. Therefore, it appears quite clear that cytokinins are likely not incorporated into tRNA directly. A model is presented (Fig. 13.6) which suggests that the free cytokinins act as a protective agent within the target cells of plants to protect the transfer-RNA molecules which contain the native cytokinin. It is suggested that there are specific nucleases which degrade only certain species of transfer RNA. The model implies that free cytokinin inhibits the nucleases through product inhibition, or possibly through some other mechanism which prevents the enzyme from attacking the tRNAs which contain the cytokinin. At present there is only a small amount of evidence to substantiate this model.