Isoprenoid cytokinins

Natural cytokinins are. / /-substituted adenine derivatives. The N6 side chain is either isoprenoid or aromatic (Figure 10), with the former occurring in greater abundance than the latter. The prenyl side chains vary in the presence or absence of a hydroxyl group and the stereoisomeric position. Common active isoprenoid cytokinins are isopentenyladenine (iP /V6-(A2-isopentenyl)adenine), trans-zeatin (tZ / / -(4-hydro.xyisopentenyl)adenine (A)-2-methyl-4-(1 //-purin-6-ylamino)but-2-en-l -ol), ax-zeatin (cZ (Z)-2-methyl-4-(l//-purin-6-ylamino)but-... 

The biosynthetic pathway producing isoprenoid cytokinins has been identified, whereas that of aromatic cytokinins is poorly characterized.363 385 Two distinct pathways for isoprenoid cytokinin biosynthesis have been described, and each pathway employs a different type of isopentenyltransferase at the initial step. The major pathway in higher plants, which is catalyzed by IPT, is conjugation of adenine nucleotide and DMAPP or 4-hydroxy-3-methyl-2-( )-butenyl diphosphate (HMBDP) (Figure 14). In the less frequently used pathway, cytokinins are formed by degradation of prenylated tRNAs. The initial prenylation reaction of tRNA is catalyzed by tRNA-isopentenyltransferase (tRNA-IPT) (Figure 14). 

Figure 15 Current model of isoprenoid cytokinin biosynthesis pathway in higher plants. tZRDP, tZ riboside 5 -diphosphate tZRTP, tZ riboside 5 -triphosphate DZRMP, DZ riboside 5 -monophosphate cZRMP, cZ riboside 5 -monophosphate ...

Two compounds common in plant metabolism are believed to be precursors of isoprenoid cytokinins in plants adenosine-5 -monophosphate (AMP) and A -isopentenylpyrophos-phate (iPP). As a final product of the mevalonate pathway, the latter substance serves also as a precursor for a wide spectrum of metabolites including some other plant hormones, as abscisic acid, gibberellins and brassinosteroids. The hypothetical scheme of reactions resulting in the formation of iPA, Z and DHZ is given in Fig. 2. The enzyme of entry into isoprenoid cytokinin formation is A -isopentenylpyrophosphate 5 -AMP-A -iso-pentenyltransferase (EC 2.5.1.8, trivially named cytokinin synthetase ). This enzyme activity was first detected in a cell-free preparation from the slime mould Dictyostelium discoideum [7,8]. Later the enzyme from higher plants (cytokinin-independent tobacco callus [9,10] and immature Zea mays kernels [11]) was described and the data were recently summarised in [12], The enzyme is very specific as far as the substrate is concerned [13,14] only the nucleotide AMP can be converted and only iPP (with a double bond in A position) may function as a side chain donor. 

The enzyme converting Z to DHZ, zeatin reductase, was characterised in Phaseolus vulgaris embryos [26]. The reduction proceeds only in the presence of NADPH and the enzyme is verj specific, with the highest affinity for Z (cZ, ZR, iPA, iPAR are not substrates, see also part 3.1.1. of this article). Taking into account that bases of isoprenoid cytokinins may represent the physiologically active forms of cytokinins [6], the conversion of Z to DHZ by zeatin reductase may prevent the loss of cytokinin activity caused by degradation of Z (but not that of DHZ, cf. part 3.1.5. of this article) by cytokinin oxidase. 

Fig. (3). Basic metabolic pathways of isoprenoid cytokinins. Enzymes participating in individual steps 1 -adenylate isopcntcnyltransfcrasc, EC 2.5.1.27 2 - cytochrome P-450 tnotiooxygcnasc, HC 1.14.13.- 3 - zeatin reductase, EC 1.3.1.69 4 - zeatin isomcrasc 5 - zcatin-O-glucosyltransferase, EC 2.4.1.203 and EC 2.4.1.215 6 - zealin-O-xylosyltamsferase, EC 2.4.2.40 7 - / -gtucosiduse, EC 3.2.1.21 8 — cytokinin-JV-glucosyltransl erose, EC 2.4.1.118 9 cytokinin dehydrogenase, EC 1.5.99.12. Dashed line indicates only predicted metabolic pathway, which have not been proved yet.

Figure 6.63 Isoprenoid cytokinin biosynthesis pathway in Arabidopsis. IPT, phosphate-isopentenyl transferase AK, adenosine kinase 1, phosphatase 2,5 -ribonucleotide phosphohydrolase 3, adenosine nucleosidase 4, purine nucleoside phosphorylase 5, zeatin cis-trans isomerase and 6, zeatin reductase.

Isoprenoids that have hormonal and signaling functions form an important group. These include steroid hormones (1 = 6) and retinoate (the anion of retinoic acid 1 = 3) in vertebrates, and juvenile hormone (1 =3) in arthropods. Some plant hormones also belong to the isoprenoids—e.g., the cytokinins, absci-sic acid, and brassinosteroids. 

An important question is Are the cytokinins present in tRNA incorporated intact into tRNA, in a manner like all other nucleoside triphosphates that are polymerized in the synthesis of tRNA, or does the presence of the cytokinins result from alkylation (transfer of isoprenoid side chain) of existing adenosine moieties already present in preformed tRNA In fact, experimental evidence has been reported for both processes (21,22). But the amounts of actual incorporation were very small, and the observed incorporation may be merely the result of transcriptional errors. Meanwhile, the evidence for cytokinin moieties in tRNA arising by alkylation of specific adenosine residues in preformed tRNA is conclusive (23). 

Terpenoids, which are also known as isoprenoids, constitute the most abundant and structurally diverse group of plant secondary metabolites, consisting of more than 40,000 different chemical structures. The isoprenoid biosynthetic pathway generates both primary and secondary metabolites that are of great importance to plant growth and survival. Among the primary metabolites produced by this pathway are phytohormones, such as gibberellic acid (GA), abscisic acid (ABA), and cytokinins the carotenoids, such as chlorophylls and plastoquinones involved in photosynthesis the ubiquinones required for respiration and the sterols that influence membrane stmcture (see also Steroid and Triterpene Biosynthesis) (Fig. 1). Monoterpenoids (CIO), sesquiterpenoids (Cl5), diterpenoids (C20), and... 

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]. 

Like all steroids, brassinosteroids derive from a single common precursor mevalonic acid. Some phytohormones are synthesized totally or in part via the isoprenoid pathway, such as absdsic acid, gibberellins and cytokinins. The importance of this biosynthetic pathway in processes involved with cell cycle regulation and tumorigenesis in mammals is well documented. Having in mind the similarities between certain regulatory systems in plants and animals the question arose whether brassinosteroids as putative plant steroid hormones would show a specific effect on plant tumor cells. 

Several important biomolecules are composed of nonterpene components attached to isoprenoid groups (often referred to as prenyl or isoprenyl groups). Examples of these biomolecules, referred to as mixed terpenoids, include vitamin E (a-tocopherol) (Figure 10.22a), ubiquinone (Figure 10.3), vitamin K, and some cytokinins (plant hormones) (Figure 11.11). 

All native cytokinins are derivatives of adenine with at least one substituent (at position). According to this substituent, these compounds may be classed into (1) isoprenoid (zeatin, A -A -isopentenyladenine and their derivatives), (2) isoprenoid-derived (dihydrozeatin and its derivatives) and (3) aromatic cytokinins. Native cytokinins and their derivatives are summarised in Fig. 1 together with abbreviations used here. 

De novo formation of isoprenoid and isoprenoid-derived cytokinins... 

Fig. 2. Hypothetical scheme of de novo formation of isoprenoid and isoprcnoid-derived cytokinins in plants modified according to [115].

To date there is no report about the biosynthesis of aromatic cytokinins. In view of the dissimilarity between the aromatic and the isoprenoid(-derived) A side chains it is likely that their biosynthetic pathways are quite different. Phenylalanine may be considered as a starting compound and benzaldehyde and/or hydroxylated benzaldehydes as immediate side chain precursors. However, the existence of some crossing-points between aromatic and isoprenoid side chain formation cannot be completely excluded. There is also the possibility that the enzymes of adenine and/or purine metabolism, which are not strictly specific, may catalyse some mutual conversions among BA-bases, nucleosides and nucleotides [81]. 

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