Cytokinins: Discovery, Measurement and Distribution | Plant Physiology

In this article we will discuss about:- 1. Discovery of Cytokinins 2. Measurement of Cytokinins 3. Distribution 4. Biogenesis and Metabolism 5. Mode of Action 6. Physiology 7. Cytokinins as RNA Constituents 8. Dormancy 9. Cytokinins and Stomata.

Contents:

1. Discovery of Cytokinins
2. Measurement of Cytokinins
3. Distribution of Cytokinins
4. Biogenesis and Metabolism of Cytokinins
5. Mode of Action of Cytokinins
6. Physiology of Cytokinins
7. Cytokinins as RNA Constituents
8. Dormancy of Cytokinins
9. Cytokinins and Stomata

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1. Discovery of Cytokinins:

These are purine derivatives especially from adenine. They are also called phytokinins. Kinetin and 6-benzylaminopurine are synthetic whereas zeatin is of natural occurrence.

Haberlandt (1913) provided circumstantial evidence about the substances from the phloem which induced cell division von Overbeek (1941) showed that coconut milk also induced cell division in embryos grown in tissue culture. Skoog (1955) isolated kinetin—an active substance which induced cell division.

It is of special significance to mention that kinetin is an artifact but subsequently cytokinin, e.g., zeatin was isolated from maize in 1964 and thereafter zeatin and other cytokinins were discovered in several species.

As compared with gibberellins and auxins, cytokinins are less mobile in the plant body. They are produced in the roots and then translocated to the leaves and shoot of the plant. This can be easily demonstrated. For instance in a tobacco plant if the roots are removed their leaves undergo senescence rapidly and the process is delayed when kinetin is supplied. The hormone seems to be translocated through xylem.

Several naturally occurring cytokinins are known and in general they are divided into two categories: amino purines and derivatives (Fig. 20-14). In the former there is a purine ring with a side chain at N₆ position. 6-furfuryl amino-purine (kinetin) belongs to this category.

Zeatin, dihydrozeatin, methylthiozeatin and DMAA are other important amino-purines. There are several synthetically prepared kinetin compounds which are prepared from urea e.g., celorophenyl-urea. Benzyl urea has been shown to antagonize the purine while phenyl urea antagonizes cytokinins.

Cytokinins stimulate growth of cotyledons of Lepidium, cucumber and soybean. It induces cell division leading to increase in fresh weight. Cytokinins also retard leaf senescence through chlorophyll retention.

Some workers have also used Sephadex LH—20 partition system for the purification of cytokinin. The purification is as high as 70 fold.

2. Measurement of Cytokinins:

Recently several papers have appeared where refined methods for the estimation of cytokinins have been given. Thin-layer chromatographic system for the separation of natural and synthetic cytokinins in addition to HPLC, novel HPLC, GC, GLC are used. However, the latter two methods have great disadvantage since cytokinins have to be derivatized before chromatographic analyses.

However, when these procedures are combined with mass spectrometry it is possible to affect selective detection and identification. Standard deuterated cytokinins have also been prepared to estimate precise recovery rates. The overall analysis takes about three days only. Very recently radioimmunosary (RIA) is being increasingly used for the quantitative analysis.

Here specificity of antigen-antibody reaction is exploited and the assay sensitivity depends on the specific radioactive of the antigen. It may be added that such methods are being used for IAA and ABA. However, for cytokinins the application of these antibodies is not conceivable. Further in most of the trial runs only synthetic benzyladenosine was used as an antigen while natural zeatin was not tried.

3. Distribution of Cytokinins:

Cytokinins have been isolated from root nodules having nitrogen fixing bacteria. In ³⁵S-labelled Rhizobium leguminosarum, etc. besides N⁶-isopenteny ladenosine, 2-methylthio-isopentenyl adenosine, 2-methylthio-ribosyl-ciszeatin have been detected in bacterial tRNA. Recent reports bring out the occurrence of side chain hydroxylated cytokinin in bacterial RNA’s.

Trans-zeatin is shown to be secreted by virulent Agrobacterium tumefaciens strain C 58. In Euglaena gracilis tRNA, cytoplasmic and chloroplast tRNA had isopentenyladenosine while cytoplasmic tRNA had additionally ribosyl-cis-zeatin exclusively and chloroplast exclusively had ribosyl-2-methylthio-zeatin.

Some workers have drawn parallelism between prokaryotes and chloroplasts with regard to the complement of cytokinin-active nucleotides in their RNA’s. Recent reports on the occurrence of ‘bryokinins’ from mosses and zeatin from gametophytes of Anemia species (Pteridophytes) are also available. From potato tubers, ‘tuber forming stimulus’, i.e., Ribosyl-cis-zeatin has been isolated. From the radish roots trans-zeatin has also been isolated.

Dihydrozeatin is optically active and a modification of the purine moiety lowers the biological activity of a cytokinin.

4. Biogenesis and Metabolism of Cytokinins:

No definite solution has been provided whether cytokinins are released intact during tRNA turnover or whether they were synthesized de novo and independent of tRNA, in lupin seeds and popular leaves, available evidences support de novo biosynthesis of free cytokinins.

In Dictyostelium disciodeum it has been demonstrated that 5′-AMP was the actual acceptor molecule for the isopentenyl group and the overall pathway for the synthesis of isopentenyladenine was as follows:

The occurrence of this reaction in higher plants remains to be verified. Recent experiments have also shown that roots are not the exclusive site of cytokinin biosynthesis. When added exogenously radioactive cytokinins undergo extensive metabolism but varied under divergent conditions.

In recent years very promising beginning has been made in studying cytokinin conjugation in cell- free systems. Incidentally serine at higher concentrations promotes leaf senescence and antagonizes cytokinin effect. In fact high serine concentration inactivates cytokinins by transforming into their 9- alanine derivatives.

Ribosylation of cytokinins basis by adenosine phoshphorylase has also been demonstrated. All the available evidences indicated that the incorporation of cytokinins into tRNA seems improbable as regards its relation to their mechanisms of action. Simultaneously cytokinins in tRNA have a positive effect on codon recognition.

5. Mode of Action of Cytokinins:

In general ‘backward’ and ‘forward’ approaches are followed to study the primary action of hormones. In the former biochemical events following hormonal treatment are recognized while in the latter hormone receptors complex is visualized. The receptor is believed to be macromolecular and proteinaceous in nature and initial-secondary reactions leading to a specific physiological responses.

In the former secondary messenger concept evolved whereas in the latter steroid hormone action be cited. In general a hormone receptor must be able to recognize hormone and generate a response. A hormone receptor must have several characteristics and in plants so far no receptor for any hormone has been distinctly identified.

However, it cannot be excluded that ribosomal binding site is involved in the recognition of the cytokinins containing tRNAs. The response of a specific tissue to an exogenously added cytokinin depends on the competence as far instance induction of bud formation in protonema of Funaria. Further of the two stages, i.e., chloronema and caulonema only the latter cells require the cytokinin.

The available reports have shown that such a response was attributed to the presence of high molecular weight caulonema specific proteins. These consider caulonema cells as target cells of cytokinin and regard them as receptor for the hormone. Reports of direct interaction of this phytohormone with membrane are also available. In some of the fungi cytokinins have been shown to interfere with cellular transport.

However, cytokinin concentration required to cause inhibition seem to be rather high. Some workers have also related cytokinin effect on K⁺ movement and on K/Ca ratio. Apparently the role of K/Ca in cytokinin mediated phenomenon is associated with an increase in K⁺ and Ca²⁺.

In some systems cytokinin and Ca²⁺ are associated with the ethylene production. Most recent works have brought out that Ca²‘ transients may regulate plant metabolism processes through Ca²⁺ dependent modulator protein enzyme like NAD kinase.

In case of hop plant cytokinin activity is attributed to zeatin and zeatin riboside in the fertilized cone and to zeatin riboside in the unfertilized cone. The level of cytokinin of the fertilized cone was nearly three times more than that of unfertilized cone. About 45% of the total cytokinin in a fertilized cone was sampled in the seeds.

Apparently increase in seed cytokinin caused rapid growth of the cone. It might have been translocated from the seeds to the cone. In apples also the seed is site of cytokinin biosynthesis and is translocated to the receptacle where it causes cell division and fruit development. Endogenous cytokinins play a significant role in cone growth.

6. Physiology of Cytokinins:

a. Cell Division:

Cytokinins are essentially needed for cell division (fig. 20-13) and cell wall formation. Instances are reported where application of cytokinins induced cell division in a mature tissue.

b. Cell Enlargement:

Addition of cytokinins to the tissue culture medium induces cell enlargement. It is effective in causing leaf enlargement in several species. It affects organ formation in coloured tissue. Prof. B.M. Johri and his students have shown that addition of 8 ppm of cytokinins to the culture medium induced shoots formation in the endosperm of misletoe.

Several studies are available to show that IAA and cytokinins induce organ formation in calli of several species. The relative proportion of the two hormones in the culture medium determines whether there will be shoot or root formation or both. However, the mode of action of the two hormones is different. For example, in pineapple IAA stimulated flowering, while zeatin induced flowering in Wolffia.

c. Accumulation of Solutes:

Cytokinins are known to accumulate solutes very actively in plants. The small size of some parasites is due to high production of cytokinins in them.

d. Prevention of Senescence:

Cytokinins when applied to detached leaves slow or prevent their senescence. The effect is in two ways: first, it prevents the formation of hydrolases, e.g., nucleases, proteases and second, it causes immobilisation of nutrients or their transport to cytokinin treated areas.

Studies by Mothes and Engelbrecht have shown younger leaves of tobacco had more of cytokinins and tended to lose less of applied14C amino acid as compared with the older leaves.

e. Enzyme Synthesis:

Kinetin has been shown to stimulate several enzymes especially those concerned with photosynthesis. The effect is at the biosynthetic level.

7. Cytokinins as RNA Constituents:

Skoog demonstrated that cytokinins were the constituents of RNA. This was especially true of tRNAs in several organisms. However, the amount of cytokinin in the tRNAs is very meagre. On an average one cytokinin is present for 20 tRNAs.

Recent studies have shown that cytokinin are essential for the function of some tRNAs but this has no relation with their hormone activity. There is also a view that cytokinin may be present as a breakdown product of tRNAs. Some workers believe that their activity is because of their presence in the RNA.

8. Dormancy of Cytokinins:

Cytokinins can also replace red light effect in seed germination of tobacco. Parasite seeds germinate more readily near the host roots because the latter exudate cytokinins.

The precise location of its action is not clear. They are known to bind to ribosomes. They possibly act on cytoplasmic ribosomes. Studies by S.P. Sen have pointed towards their action on nuclear ribosomes as well. Fox and Cherry have proposed that tRNA lacking isopentenyl side chain in the adenine next to anticodon are not active.

Thus cytokinins may be determining the conformation of anticodon. There is every likelihood that cytokinins influence growth through two forms, one in the free state and the other one incorporated inside tRNAs. The free cytokinins may also be protecting tRNAs from ribonucleases or may even help in the synthesis of fresh ribosomal RNA.

Morphogenesis:

The most striking response to cytokinins is the re-differentiation of certain plant tissue cultures to form organs. With auxin they regulate morphogensis. In many plant callus, ratio of auxin to cytokinin affects root and shoot formation. Crown gall tissues provide some evidences for the control of growth and differentiation in plant tissue cultures.

9. Cytokinins and Stomata:

In the isolated epidermal peels system, cytokinin exhibit very little effect on stomata aperture. However, application of cytokinin to the whole leaf of a graminaceous member increases transpiration.