Let us make an in-depth study of the kinetin and cytokinins in plants. After reading this article you will learn about 1. Discovery and Chemical Nature of Kinetin and Cytokinins 2. Zeatin 3. Other Natural Cytokinins 4. Cytokinins in t-RNA 5. Biosynthesis of Zeatin & Other Natural Cytokinins 6. Deactivation of Cytokinins 7. Distribution and Transport of Cytokinins in Plants and 8. Mechanism of Cytokinin Action.
Discovery and Chemical Nature of Kinetin and Cytokinins :
The discovery of Kinetin is comparatively more recent. Its credit goes to Miller et al (1955) who were working in Prof. Skoog’s lab. at the University of Wisconsin on the growth of tobacco pith callus in culture and wanted it to grow indefinitely.
They added various growth substances, nutrients, vitamins etc. into the culture medium but failed till they noticed an old bottle of DNA kept for several years in their lab. They added the contents of that bottle to the culture medium and observed that the tobacco pith callus could grow for longer periods. They obtained similar results with Yeast extract. But they did not get positive results with fresh DNA and thought the active substance to be some degradation product of DNA.
They isolated this substance by autoclaving (heating under pressure) the DNA which had been stored for long. It could easily be precipitated by silver salts and was soluble in 90% alcohol, indicating that possibly it was a purine compound. Later on, they identified it as 6-furfurylaminopurine. Because of its specific effect on cytokinesis i.e., cell division, it was called as kinetin (Fig. 17.21).
Although kinetin has profound influences in inducing cell division, still it has not been isolated from any plant. But, certain substances which show kinetin like activity have in fact been isolated from a variety of higher plants. These substances are collectively called as cytokinins. There is now sufficient evidence to show that cytokinins do occur in plants and regulate growth and hence, they are also considered as natural plant growth hormones.
Some of the very important and commonly known naturally occurring cytokinins are as follows:
Zeatin:
Zeatin is the most abundant and widely distributed natural cytokinin in higher plants and in some bacteria. Although this cytokinin was known earlier but it was obtained in pure crystalline form in 1963 by Letham from immature corn grains and named as Zeatin. It was identified as 6-(4-hydroxy-3-methylbut-trans-2-enyl) amino purine by Letham et al (1964) and was synthesized by Shaw and Wilson (1964).
Zeatin exhibits strong kinetin like activity in stimulating plant cell to divide in presence of auxin in culture media.
i. Zeatin resembles kinetin in molecular structure because both are adenine or amino purine derivatives.
ii. Zeatin is remarkably more active than any other cytokinin probably because of the presence of a highly reactive allylic-OH group in its side chain.
iii. Due to the presence of a double bond in its side chain, zeatin can exist either in trans or cis form (Fig. 17.22). These two forms can be interconverted in the presence of the enzyme zeatin isomerase.
iv. Transzeatin is predominant and biologically most active form of this hormone. Cis-zeatin may also play a biological role. Cis-form predominates in cytokinins which are bound to t-RNAs.
Other Natural Cytokinins:
Apart from zeatin, some other substituted amino purines have been isolated from higher plants and some bacteria which are also considered as natural cytokinins. These are, dihydrozeatin (DZ) and N6-(Δ2-isopentenyl) adenine (or ip) which differ from zeatin in nature of their side chain (Fig. 17.23).
i. Numerous derivatives of these three cytokinins (Z, diHZ and iP) have been identified in plant extracts.
ii. Zeatin and other natural cytokinins commonly occur as ribosides or ribotides in which ribose sugar is attached to N at 9th position of the purine ring. They may also occur as glucosides where the glucose molecule is attached to N at 3, 7 or 9th position of purine ring or to oxygen (O) in side chain of zeatin or dihydrozeatin. These bound forms of cytokinins are however, not biologically active: (N3-glucosides are exceptional in being biologically active in bioassays).
iii. Zeatin and other natural cytokinins are hormonally active only in their free base forms.
(Cytokinins have also been isolated from algae, mosses, ferns and conifers. Some plant pathogenic bacteria, nematodes and insects also secrete cytokinins or they induce plant cells to biosynthesize cytokinins and other growth hormones).
Cytokinin Antagonists:
Some chemical compounds are known to act as cytokinin antagonists. Example of one such compounds is 3-Methyl -7-(3-methylbutylamino) pyrazolo [4, 3-D] pyrimidine.
Cytokinins in t-RNA:
In 1966 Zachau et al identified a cytokinin 2iPA as a constituent of two serine t-RNA species from brewer’s yeast and showed this cytokinin to be adjacent to the 3′ end of the anticodon in both the species. Apart from yeast, cytokinins have now been found in t-RNA preparations from a wide variety of organisms such as bacteria including E .coli, animals including man and higher plants viz., corn seedlings, immature corn kernels, frozen peas, tobacco callus tissue and wheat germ.
It has been suggested by various workers that the association of a particular cytokinin with anticodon of the t-RNA molecule might have a bearing on codon-anticodon interaction between t-RNA and m-RNA during protein synthesis. For instance, in case of yeast serine t- RNA the chemical modification of the cytokinin (associated with the latter) had no effect on the acceptance of serine by the t-RNA but interfered with the subsequent binding of the charged t-RNA to the m-RNA ribosomal complex.
Synthetic Cytokinins:
Some synthetic chemical compounds which show cytokinin activity but have not been isolated from plants are known. Benzyl adenine (BA) is one such example. Although there are a few reports of this compound in plants but it is uncommon in plants and is largely a synthetic cytokinin. Another synthetic cytokinin is thidiazuron that is used commercially as defoliant and a herbicide.
Biosynthesis of Zeatin & Other Natural Cytokinins:
Biosynthesis of free cytokinins has been shown schematically in Fig. 17.24.
i. Cytokinins are synthesized from adenosine monophosphate (AMP) and isopentenylpyrophosphate (∆2-iPP) by condensation reaction that is catalysed by the enzyme isopentenyl transferase. The product of this condensation is N6-(∆2-isopentenyl)- adenosine- 5′-monophosphate (i.e., [9R-5-P] iP) which is supposed to be precursor to all other natural cytokinins.
(The isopentenyl pyrophosphate is in-fact transferred from dimethylallyl pyrophosphate (DMAPP) to AMP. The enzyme isopentenyl transferase is specific to nucleotide only. It will not bind with either adenine or adenosine. This enzyme has been isolated from slime mold Dictyostelium discoideum, tobacco callus tissue and crown gall tissue).
i. The [9R-5’-P] iP is readily dephosphorylated to yield N6-(Δ2– isopentenyl)- adenosine.
ii. Ribose sugar is now removed from N6-(Δ2– isopentenyl)- adenosine, so that N6-(Δ2– isopentenyl)- adenine (i.e., iP) is formed.
iii. Isopentenyl side chain of iP is now hydroxylated to form free zeatin.
Alternatively, [9R-5-P] iP may be hydroxylated directly to give 9-ribosyl-zeatin-5′- phosphate (i.e. [9R-5’P] Z). The phosphate group and then the ribose sugar are removed from [9R- 5’P]Z in sequence to form free zeatin.
iv. Reduction of the double bond in isopentenyl side chain of zeatin would give rise to dihydrozeatin (diHZ).
As mentioned earlier, the cytokinins are also found in t-RNA in wide variety of organisms from bacteria to man. However free cytokinins in cells do not arise simply by hydrolysis of cytokinins containing t-RNAs. Experimental evidences have suggested and it is generally believed that there is de novo synthesis of free cytokinins in cells.
Deactivation of Cytokinins:
Like other growth hormones, sufficient levels of cytokinins in plant required for regulation of plant growth are maintained not only by their synthesis but also by their destruction or inactivation. There are two main routes for inactivation of cytokinins in plants, (i) by conjugation and (ii) by oxidation.
1. By conjugation (Reversible or irreversible):
In this method, cytokinin level can be regulated by conjugation of cytokinin with either glucose or amino acids which may be reversible or irreversible depending upon the nature of conjugation.
i. In some plants, cytokinins can be glucosylated through N at 3rd, 7th or 9th positions on the purine ring. These N-glucosides are very stable and are not readily hydrolysed to give active free bases. Both 7-and 9-glucosides are biologically inactive. Therefore, such modifications are generally irreversible. (The N3-glucoside is however, active in bio- assays).
ii. Glucosylation of cytokinins through side chain-OH group (i.e., O-glucosylation) is also very common method of inactivation of cytokinins in plants. O-glucosides are resistant to oxidation by cytokinin oxidases. Only transzeatin and dihydrozeatin bases can be O-glucosylated by specific enzymes but not their riboside or ribotide forms. However, conjugations at the side chain can be removed by glucosidases to yield free cytokinins. Therefore, such modifications are generally reversible. (In some cases, xylose instead of glucose may be involved in conjugation forming O-xylosides).
iii. The amino acid alanine can also be conjugated with cytokinin through N at 9th position of purine ring forming inactive lupinic acid. Like N-glucosides, this conjugation is also very stable and generally irreversible.
2. By Oxidation (Irreversible):
In many plant tissues, cytokinins are irreversibly inactivated (rather destroyed) by the enzyme cytokinin oxidase which cleaves the isopentenyl side chain of cytokinins (Fig. 17.25). Both trans and cis forms of zeatin, zeatin riboside, iP and their N-glucosides are the substrates for this enzyme. However, O-glucoside derivatives are not the substrates.
Distribution and Transport of Cytokinins in Plants:
Unlike auxins and gibberellins, the cytokinins are not widely distributed in different plant parts. There are evidences to believe that cytokinins are synthesized in roots especially during seedling stage and trans located to shoots and leaves of the plant. Highest concentrations of cytokinins are observed in root and shoot tips.
The movement of cytokinins in plant too is not as readily as those of auxin and gibberellin. Cytokinins have been found in xylem exudate and appear to be trans located through xylem. It is noteworthy that in many experiments it has been found that when cytokinin is applied to a leaf or a particular tissue, it does not migrate but stays where it was applied.
(The cytokinins are transported in their bound form as ribosides in plant. A signal from shoot regulates the transport of zeatin ribosides from roots. However, the identity of this signal remains to be determined).
Mechanism of Cytokinin Action:
The precise mechanism of the action of cytokinins at cellular and molecular levels in plants is still unknown and our knowledge about these is fragmentary. However, in recent years some light has been thrown by scientists on the mechanism of action of cytokinins.
In Arabidopsis, a cytokinin receptor has been identified which is a trans membrane protein called as CRE1 and is similar to bacterial two component sensor histidine kinases. CRE1 is probably a dimer, each polypeptide of which consists of three domains,
(i) An extracellular CHASE domain towards the amino terminus,
(ii) A middle His (Histidine) kinase domain and
(iii) A receiver domain towards the carboxyl terminus of the polypeptide (Fig. 17.26).
Besides this, two other hybrid sensor histidine kinases in Arabidopsis called AHK2 and AHK3 with CHASE domains may also be cytokinin receptors.
Some important events of cytokinin signalling are shown in Fig. 17.26. and are as follows :
(i) Cytokinin binds to extracellular CHASE domain of the receptor (CRE1 /AHK2/AHK3) present on plasma membrane.
(ii) The middle Histidine kinase domain of the receptor is activated (i.e., phosphorylated) by ATP and the phosphate is transferred to the aspartate residue (D) on fused receiver domain of the receptor.
(iii) From receiver domain, the phosphate (P) is transferred to a conserved histidine residue located in an AHP protein (i.e., Arabidopsis Histidine Phosphotransfer protein) The phosphorylated AHP protein moves to nucleus.
(iv) In nucleus, the phosphorylated AHP protein transfers its phosphate group to an aspartate residue present in receiver domain of type B ARR (Arabidopsis Response Regulator) that consists of a receiver domain and an additional transcription factor domain or output domain).
Type B ARRs may interact with other effectors leading to cytokinin responses and/or,
(v) The phosphorylated B type ARR activates its own output domain (i.e., transcription factor domain) and induces transcription of genes which encode type A ARRs (ARRs which consist of only a receiver domain).
(vi) Type A ARRs may also be phosphorylated by phosphorylated AHP (See step iv).
(vii) Phosphorylated type A ARRs may now interact with other effectors leading to cytokinin responses.