Partial Purification of a cis-trans-Isomerase of Zeatin from Immature Seed of Phaseolus vulgaris L

Investigation of the conversion of exogenous cis-zeatin to trans-zeatin in immature seeds of Phaseolus vulgaris L. led to the isolation of a cis-trans-isomerase from the endosperm. The enzyme was purified more than 2000-fold by chromatography on a series of fast protein liquid chromatography (anion exchange, gel filtration, and hydrophobic interaction) and concanavalin A columns. The enzymic reaction favors conversion from the cis to the trans form and requires flavin, light, and dithiothreitol. cis-Zeatin riboside is also a substrate for the enzyme. Retention on the concanavalin A column indicated that the enzyme is a glycoprotein. The enzyme was stable for at least 8 weeks when stored at -80[deg] C. The occurrence of cis-trans-isomerization suggests that cis-zeatin and cis-zeatin riboside formed by tRNA degradation could be precursors of biologically active cytokinins.     

O-glucosylation of cis-zeatin in maize. Characterization of genes,enzymes, and endogenous cytokinins

trans-Zeatin is a major and ubiquitous cytokinin in higher plants. cis-Zeatin has traditionally been viewed as an adjunct with low activity and rare occurrence. Recent reports of cis-zeatin and its derivatives as the predominant cytokinin components in some plant tissues may call for a different perspective on cis-isomers. The existence of a maize (Zea mays) gene (cisZOG1) encoding an O-glucosyltransferase specific to cis-zeatin (R.C. Martin, M.C. Mok, J.E. Habben, D.W.S. Mok [2001] Proc Natl Acad Sci USA 98: 5922-5926) lends further support to this view. Results described here include the isolation of a second maize cisZOG gene, differential expression of cisZOG1 and cisZOG2, and identification of substantial amounts of cis-isomers in maize tissues. The open reading frame of cisZOG2 has 98.3% identity to cisZOG1 at the nucleotide level and 97.8% at the amino acid level. The upstream regions contain common and unique segments. The recombinant enzymes have similar properties, K(m) values of 46 and 96 microM, respectively, for cis-zeatin and a pH optimum of 7.5. Other cytokinins, including N(6)-(delta(2)-isopentenyl)adenine, trans-zeatin, benzyladenine, kinetin, and thidiazuron inhibited the reaction. Expression of cisZOG1 was high in maize roots and kernels, whereas cisZOG2 expression was high in roots but low in kernels. cis-Zeatin, cis-zeatin riboside, and their O-glucosides were detected in all maize tissues, with immature kernels containing very high levels of the O-glucoside of cis-zeatin riboside. The results are a clear indication that O-glucosylation of cis-zeatin is a natural metabolic process in maize. Whether cis-zeatin serves as a precursor to the active trans-isomer or has any other unique function remains to be demonstrated.     

Evidence in support of biosynthesis de novo of free cytokinins

The four cytokinins in the tRNA from Lupinus luteus L. seeds have been purified and identified as ribosyl-cis-zeatin, 2-methylthio-ribosylzeatin, ( ²-isopentenyl)adenosine and 2-methylthio-N⁶-( ²-isopentenyl)adenosine. These structures have been assigned on the basis of their chromatographic mobilities and the spectroscopic data of the parent materials and their silylated derivatives. The tRNA isolated from Populus x robusta Schneid. leaves contained four cytokinins with identical chromatographic properties to those identified in Lupinus luteus seed tRNA. No evidence was obtained for the presence, in tRNA, of the naturally occurring free cytokinins identified in these plant species, dihydrozeatin (Lupinus luteus) and N⁶-(2-hydroxybenzyl)adenosine (Populus x robusta). This is evidence in support of the possibility that free cytokinins can arise by biosynthesis de novo and are not exclusively by-products released intact during tRNA turnover.     

Transfer RNA Is the Source of Extracellular Isopentenyladenine in a Ti-Plasmidless Strain of Agrobacterium tumefaciens

Even in the absence of the classical Ti plasmid-encoded cytokinin biosynthetic genes ipt and tzs, Agrobacterium tumefaciens strains still release significant amounts of the cytokinin isopentenyladenine (iP) into the culture medium (R.W. Kaiss-Chapman and R.O. Morris [1977] Biochem Biophys Res Commun 76: 453-459). A potential source of the iP is isopentenylated transfer RNA (tRNA), which, in turn, is synthesized by the activity of tRNA:isopentenyltransferase encoded by the bacterial miaA gene. To determine whether secreted iP had its origin in isopentenylated tRNA, a miaA- deletion/insertion mutant was prepared and reconstructed in Agrobacterium tumefaciens in vivo. The mutant no longer possessed tRNA:isopentenylation activity and no longer released iP into the extracellular medium. Transfer RNA therefore makes a small but significant contribution to the total amount of cytokinin normally secreted by Agrobacterium strains. tRNA-mediated synthesis may also account for cytokinin production by other plant-associated bacteria, such as Rhizobia, that have been reported to secrete similarly low levels of nonhydroxylated cytokinins.     

Immunochemical identification of a tRNA-independent cytokinin-like compound in Salmonella typhimurium

Chemical and immunological characterization of Salmonella typhimurium cell extracts indicates that this organism produces a molecule which closely resembles the plant growth regulator, cytokinin. Alcohol-soluble cationic ultraviolet-absorbing material was fractionated by reverse-phase HPLC using gradient conditions optimized previously for modified nucleoside separation. A single hydrophobic compound was identified in the cytokinin region of the gradient, and limited quantities of the compound were prepared by HPLC fractionation of crude extracts. The compound demonstrated significant activity in a radioimmunoassay for cytokinins which detects N6-isopentenylated adenine derivatives. Boronate affinity chromatography indicated the compound is likely to be ribosylated and therefore a nucleoside. These and other tests indicate the compound has the most notable structural characteristics of a cytokinin. Spectral analysis and chromatographic comparison with cytokinin standards indicate the compound also has some unique structural features. Presence of the compound in extracts of an S. typhimurium mutant blocked for synthesis of tRNA-derived cytokinins excluded tRNA as a source for the compound and implicates existence of a tRNA-independent pathway for cytokinin biosynthesis in this bacterial species.     

Two cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, differ in their ligand specificity in a bacterial assay

Strains of Escherichia coli that express two different cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, were used to study the relative sensitivity of these receptors to various cytokinins. Both receptors were most sensitive to the bases of the isoprenoid-type cytokinins trans-zeatin and isopentenyladenine but differed significantly in the recognition of other cytokinin compounds. In particular, CRE1/AHK4 recognized at 1 microm concentration only trans-zeatin while AHK3 recognized cis-zeatin and dihydrozeatin as well, although with a lower sensitivity. Similarly, CRE1/AHK4 was not activated by cytokinin ribosides and ribotides, but AHK3 was. Comparisons using the ARR5::GUS fusion gene as a cytokinin reporter in Arabidopsis showed similar relative degrees of responses in planta, except that cytokinins with aromatic side chains showed much higher activities than in the bacterial assay. These results indicate that the diverse cytokinin compounds might have specific functions in the numerous cytokinin-regulated processes, which may depend in turn on different receptors and their associated signalling pathways. The importance of precise control of local concentrations of defined cytokinin metabolites to regulate the respective downstream event is corroborated.     

Subcellular localization of cytokinins in transfer ribonucleic Acid

Evidence on the localization of cytokinins in chloroplast tRNA was obtained by comparison of Euglena gracilis var. bacillaris light-grown and dark-grown wild type cultures and chloroplast-bleached mutant strains. The several cytokinins characteristic of tRNA were separated by Sephadex LH-20 column chromatography of the hydrolysates and were quantitatively determined by tobacco bioassays of the eluates. The results indicate that 6-(3-methyl-2-butenylamino)-9-beta-d-ribofuranosylpurine (i(6) A) is formed in both the cytoplasmic and chloroplast tRNA, whereas 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9-beta-d- ribofurano-sylpurine (c-io(6)A) is produced mainly in the cytoplasmic tRNA and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-beta-d- ribofurano-sylpurine (ms(2)io(6)A) is localized exclusively in chloroplast tRNA. The restriction of the methiolation reaction to the chloroplast is supported by results of radioisotope experiments showing that (35)S-labeled MgSO(4) is incorporated into ms(2)io(6)A in the wild type cultures, but not in the chloroplast-bleached mutant strains.     

Identification of a tRNA isopentenyltransferase gene from Arabidopsis thaliana

The tRNA of most organisms contain modified adenines called cytokinins. Situated next to the anticodon, they have been shown to influence translational fidelity and efficiency. The enzyme that synthesizes cytokinins on pre-tRNA, tRNA isopentenyltransferase (EC 2.5.1.8), has been studied in micro-organisms like Escherichia coli and Saccharomyces cerevisiae, and the corresponding genes have been cloned. We here report the first cloning and functional characterization of a homologous gene from a plant, Arabidopsis thaliana. Expression in S. cerevisiae showed that the gene can complement the anti-suppressor phenotype of a mutant that lacks MOD5, the intrinsic tRNA isopentenyltransferase gene. This was accompanied by the reintroduction of isopentenyladenosine in the tRNA. The Arabidopsis gene is constitutively expressed in seedling tissues.     

Cytokinin biosynthesis and interconversion

To maintain hormone homeostasis, the rate of cytokinin biosynthesis, interconversion, and degradation is regulated by enzymes in plant cells. Cytokinins can be synthesized via direct (de novo) or indirect (tRNA) pathways. In the de novo pathway, a cytokinin nucleotide is synthesized from 5'-AMP and isopentenyl pyrophosphate; a key enzyme which catalyzes this synthesis has been isolated from plant tissues, slime mold, and some microorganisms. Studies on the in vitro synthesis of the isopentenyl side chain of cytokinin in tRNA demonstrated that the isopentenyl group was derived from mevalonate, and turnover of the cytokinin-containing tRNA may serve as a minor source of free cytokinins in plant cells. The interconversion of cytokinin bases, nucleosides and nucleotides is a major feature of cytokinin metabolism; and enzymes that regulate the interconversion have been identified. The N⁶-side chain and purine moiety of cytokinins are often modified and some of the enzymes involved in the modifications have been isolated. Most of the cytokinin metabolites have been characterized but very few enzymes regulating their metabolism have been purified to homogeneity. It remains a significant challenge to isolate plant genes involved in the regulation of cytokinin biosynthesis, interconversion and degradation.     

Gamma-irradiated plant callus tissue: Cytokinins from transfer ribonucleic acid

Summary Callus tissue ofHaworthia mirabilis Haw. was irradiated with⁶⁰Co gamma rays. tRNA was isolated, hydrolyzed enzymatically, and cytokinin-active ribonucleosides were separated by Sephadex LH-20 column chromatography and assayed with the tobaccocallus cytokinin bioassay. Three cytokinins were detected in tRNA from irradiated tissue, two of which chromatographed with zeatin riboside and N⁶-(Δ²-isopentenyl)adenosine. The third cytokinin-active ribonucleoside was retained longer than the above compounds on the Sephadex column and may be 2-methylthio-N⁶-(Δ²-isopentenyl)adenosine. Two cytokinins were detected in tRNA from nonirradiated tissue—those chromatographed with zeatin riboside and N⁶-(Δ²-isopentenyl)adenosine. Relationships between cytokinins from tRNA and free cytokinins found in tissue earlier are discussed. This is paper 78-10-124 of the Kentucky Agricultural Experiment Staton and is published with approval of the Director.     

The role of auxins and cytokinins in the mutualistic interaction between Arabidopsis and Piriformospora indica

Arabidopsis growth and reproduction are stimulated by the endophytic fungus Piriformospora indica. The fungus produces low amounts of auxins, but the auxin levels and the expression of auxin-regulated genes are not altered in colonized roots. Also, mutants with reduced auxin levels (ilr1-1, nit1-3, tfl2, cyp79 b2b3) respond to P. indica. However, the fungus rescues the dwarf phenotype of the auxin overproducer sur1-1 by converting free auxin into conjugates, which also results in the downregulation of the auxin-induced IAA6 and the upregulation of the P. indica-induced LRR1 gene. The fungus produces relatively high levels of cytokinins, and the cytokinin levels are higher in colonized roots compared with the uncolonized controls. trans-Zeatin cytokinin biosynthesis and the CRE1/AHK2 receptor combination are crucial for P. indica-mediated growth stimulation, while mutants lacking cis-zeatin, impaired in other cytokinin receptor combinations, or containing reduced cytokinin levels respond to the fungus. Since root colonization is not affected in the cytokinin mutants, we propose that cytokinins are required for P. indica-induced growth promotion. Finally, a comparative analysis of the phytohormone mutants allows the conclusion that the response to P. indica is independent of the architecture and size of the roots.     

Cytokinins in the bryophyte Physcomitrella patens: analyses of activity, distribution, and cytokinin oxidase/dehydrogenase overexpression reveal the role of extracellular cytokinins

Ultra-performance liquid chromatography-tandem mass spectrometry was used to establish the cytokinin profile of the bryophyte Physcomitrella patens (Hedw.) B.S.G.; of 40 analyzed cytokinins, 20 were detected. cis-Zeatin-riboside-O-glucoside, N(6)-(Delta(2)-isopentenyl)adenosine-5'-monophosphate (iPRMP), and trans-zeatin-riboside-O-glucoside were the most abundant intracellular cytokinins. In addition, the aromatic cytokinins N(6)-benzyladenosine (BAR), N(6)-benzyladenine, meta-, and ortho-topolin were detected. Unexpectedly, the most abundant extracellular cytokinin was the nucleotide iPRMP, and its identity was confirmed by quadrupole time-of-flight mass spectrometry. The effects of overexpressing a heterologous cytokinin oxidase/dehydrogenase (CKX; EC 1.4.3.18/1.5.99.12) gene (AtCKX2 from Arabidopsis [Arabidopsis thaliana]) on the intracellular and extracellular distribution of cytokinins was assessed. In cultures of CKX-transformed plants, ultra-performance liquid chromatography-tandem mass spectrometry measurements showed that there were pronounced reductions in the extracellular concentrations of N(6)-(Delta(2)-isopentenyl)adenine (iP) and N(6)-(Delta(2)-isopentenyl)adenosine (iPR), but their intracellular cytokinin concentrations were only slightly affected. In vitro and in vivo measured CKX activity was shown to be strongly increased in the transformants. Major phenotypic changes observed in the CKX-overexpressing plants included reduced and retarded budding, absence of sexual reproduction, and abnormal protonema cells. In bud-induction bioassays with wild-type Physcomitrella, the nucleotides iPRMP, trans-zeatin-riboside-5'-monophosphate, BAR monophosphate, and the cis-zeatin forms cZ and cZR had no detectable effects, while the activities displayed by other selected cytokinins were in the following order: iP > tZ > N(6)-benzyladenine > BAR > iPR > tZR > meta-topolin > dihydrozeatin > ortho-topolin. The results on wild type and CKX transgenics suggest that extracellular iP and iPR are the main cytokinins responsible for inducing buds in the bryophyte Physcomitrella. Cytokinin profile is discussed regarding the evolution of cytokinin biosynthetic pathways.     

Transport of cytokinins mediated by purine transporters of the PUP family expressed in phloem,hydathodes, and pollen of Arabidopsis

Nucleobases and derivatives like cytokinins and caffeine are translocated in the plant vascular system. Transport studies in cultured Arabidopsis cells indicate that adenine and cytokinin are transported by a common H+-coupled high-affinity purine transport system. Transport properties are similar to that of Arabidopsis purine transporters AtPUP1 and 2. When expressed in yeast, AtPUP1 and 2 mediate energy-dependent high-affinity adenine uptake, whereas AtPUP3 activity was not detectable. Similar to the results from cell cultures, purine permeases (PUP) mediated uptake of adenine can be inhibited by cytokinins, indicating that cytokinins are transport substrates. Direct measurements demonstrate that AtPUP1 is capable of mediating uptake of radiolabeled trans-zeatin. Cytokinin uptake is strongly inhibited by adenine and isopentenyladenine but is poorly inhibited by 6-chloropurine. A number of physiological cytokinins including trans- and cis-zeatin are also efficient competitors for AtPUP2-mediated adenine uptake, suggesting that AtPUP2 is also able to mediate cytokinin transport. Furthermore, AtPUP1 mediates transport of caffeine and ribosylated purine derivatives in yeast. Promoter-reporter gene studies point towards AtPUP1 expression in the epithem of hydathodes and the stigma surface of siliques, suggesting a role in retrieval of cytokinins from xylem sap to prevent loss during guttation. The AtPUP2 promoter drives GUS reporter gene activity in the phloem of Arabidopsis leaves, indicating a role in long-distance transport of adenine and cytokinins. Promoter activity of AtPUP3 was only found in pollen. In summary, three closely related PUPs are differentially expressed in Arabidopsis and at least two PUPs have properties similar to the adenine and cytokinin transport system identified in Arabidopsis cell cultures.     

Endogenous cytokinins in the ribosomal RNA of higher plants

Endogenous cytokinin-active ribonucleosides were isolated from the rRNA and tRNA of pea epicotyls (Pisum sativum L., var Alaska) and of wheat germ (Triticum aestivum). The RNA preparations were analyzed for cytokinins by enzymic hydrolysis, ethyl acetate extraction, and Sephadex LH-20 fractionation in several solvents. Tentative identification of the cytokinins was based on cochromatography with synthetic cytokinin standards in several systems and on activity in the tobacco bioassay. Both the rRNA and tRNA from 10 day old pea epicotyls contained ribosylzeatin, isopentenyladenosine, and 2-methylthioribosylzeatin. The latter compound was the most active fraction in the pea rRNA, but was the least active fraction in the tRNA, where isopentenyladenosine activity was predominant. The 2-methylthioribosylzeatin from pea rRNA was identified by gas chromatography-mass spectrometry. Wheat germ rRNA contained cis and trans ribosylzeatin and 2-methylthioribosylzeatin. The tRNA contained isopentenyladenosine in addition. The specific cytokinin activity (activity per A₂₆₀ unit) of the tRNA was over forty times that of the rRNA. Significant contamination of the rRNA preparations by cytokinin-containing tRNA is considered unlikely on the basis of quantitative differences in the cytokinin content of the rRNA and tRNA preparations, electrophoretic analysis of rRNA purity and cytokinin analysis of fractionated oligonucleotide digests.     

Transfer RNA, a Possible Supplier of Free Cytokinins, Ribosyl-cis-zeatin and Ribosyl-2-methylthiozeatin: Quantitative Comparison between Free and Transfer Cytokinins in Various Tissues of the Hop Plant

Cytokinins in both free pool and tRNAs have been identifiedand quantified in the unfertilized cone, fertilized cone, seed,cotyledon, epicotyl and root of the hop plant (Humulus lupulusL. cv. Shinshu-wase) on the basis of combined gas chromatography-massspectrometry and combined gas chromatography-selected ion currentmonitoring analysis. Based on the quantitative comparison betweenfree pool cytokinins and tRNA cytokinins, it is suggested thatribosyl-cis-zeatin and ribosyl-2-methylthiozeatin might be derivedfrom tRNA catabolism and that the level of ribosyl-trans-zeatinmight be controled independently of the tRNA catabolism. ¹ Present address: Taisho Pharmaceutical Co. Ltd., Yoshini-cho,Omiya-shi, Saitama 330, Japan. (Received December 1, 1981; Accepted February 9, 1982)     

Biochemical characteristics and ligand-binding properties of Arabidopsis cytokinin receptor AHK3 compared to CRE1/AHK4 as revealed by a direct binding assay

The cytokinin receptor AHK3 of Arabidopsis thaliana plays a predominant role in shoot development. A study of the hormone-binding characteristics of AHK3 compared with the mainly root-confined receptor CRE1/AHK4 has been accomplished using a live-cell binding assay on transgenic bacteria expressing individual receptor proteins. Both receptors bound trans-zeatin (tZ) with high affinity. Scatchard analysis showed a linear function corresponding to an apparent K(D) of 1-2 nM for the AHK3 receptor-hormone complex, which is close to the K(D) (2-4 nM) for the CRE1/AHK4 receptor-hormone complex. The specific binding of tZ to both receptors was pH dependent, AHK3 being more sensitive to pH changes than CRE1/AHK4. Hormone binding was reversible, at least for the bulk of (3)H-zeatin, and influenced by monovalent cations, while divalent cations (Ca(2+), Mg(2+), Mn(2+)) at physiological concentrations had no significant effect. AHK3 differed significantly from CRE1/AHK4 in relative affinity to some cytokinins. AHK3 had an approximately 10-fold lower affinity to isopentenyladenine (iP) and its riboside, but a higher affinity to dihydrozeatin than CRE1/AHK4. For AHK3, cytokinin ribosides (tZR, iPR) and cis-zeatin had true binding activity, although lower than that of tZ. The phenylurea-derived cytokinin thidiazuron was a strong competitor and bound to the same site as did adenine-derived cytokinins. The inhibitor of cytokinin action butan-1-ol had little effect on cytokinin-receptor complex formation. The revealed properties of AHK3 suggest its specific function in root-to-shoot communication.     

Topolins and hydroxylated thidiazuron derivatives are substrates of cytokinin O-glucosyltransferase with position specificity related to receptor recognition

Glucosides of trans-zeatin occur widely in plant tissues, formed either by O-glucosylation of the hydroxylated side chain or N-glucosylation of the purine ring structure. O-Glucosylation is stereo-specific: the O-glucosyltransferase encoded by the Phaseolus lunatus ZOG1 gene has high affinity for trans-zeatin as the substrate, whereas the enzyme encoded by the maize (Zea mays) cisZOG1 gene prefers cis-zeatin. Here we show that hydroxylated derivatives of benzyladenine (topolins) are also substrates of ZOG1 and cisZOG1. The m-OH and o-OH derivatives are the preferred substrate of ZOG1 and cisZOG1, respectively. Among the hydroxylated derivatives of thidiazuron tested, the only enzyme/substrate combination resulting in conversion was cisZOG1/(o-OH) thidiazuron. The abilities of these cytokinins to serve as substrates to the glucosyltransferases were in a large part correlated with their biological activities in the P. lunatus callus bioassay, indicating that there may be similarities between cytokinin-binding sites on the enzymes and cytokinin receptors. Further support for this interpretation is provided by cytokinin recognition studies involving the Arabidopsis (Arabidopsis thaliana) CRE1/WOL/AHK4 and maize ZmHK1 receptors. The AHK4 receptor responded to trans-zeatin and m-topolin, while the ZmHK1 receptor responded also to cis-zeatin and o-topolin. Three-dimensional molecular models of the substrates were applied to explain the results.     

Mass spectrometry and chromatography of t-butyldimethylsilyl derivatives of cytokinin bases

Di-(t-butyldimethylsilyl) derivatives of the cytokinin bases zeatin, cis-zeatin, and dihydrozeatin may be prepared quantitatively in the presence of dimethylaminopyridine. These derivatives have good gas chromatographic properties and are very suitable for gas chromatography-mass spectrometry analysis of cytokinin bases. The t-butyldimethylsilyl (tBuDMS) group at N-9 may be selectively hydrolyzed and the resulting mono-O-silyl derivatives are sufficiently stable to be subjected to thin-layer chromatography and high-performance liquid chromatography. The mass spectral fragmentation of the mono- and di-tBuDMS derivatives of adenine, zeatin, cis-zeatin, and dihydrozeatin and also of the mono-tBuDMS derivatives of N6-isopentenyladenine and 6-benzylaminopurine have been rationalized. The 9-tBuDMS moiety was characterized by an elimination of isobutene (M-56) and of isobutene plus a methyl radical (M-56-15).     

Distinct isoprenoid origins of cis- and trans-zeatin biosyntheses in Arabidopsis

Plants produce the common isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP) through the methylerythritol phosphate (MEP) pathway in plastids and the mevalonate (MVA) pathway in the cytosol. To assess which pathways contribute DMAPP for cytokinin biosynthesis, metabolites from each isoprenoid pathway were selectively labeled with (13)C in Arabidopsis seedlings. Efficient (13)C labeling was achieved by blocking the endogenous pathway genetically or chemically during the feed of a (13)C labeled precursor specific to the MEP or MVA pathways. Liquid chromatography-mass spectrometry analysis demonstrated that the prenyl group of trans-zeatin (tZ) and isopentenyladenine is mainly produced through the MEP pathway. In comparison, a large fraction of the prenyl group of cis-zeatin (cZ) derivatives was provided by the MVA pathway. When expressed as fusion proteins with green fluorescent protein in Arabidopsis cells, four adenosine phosphate-isopentenyltransferases (AtIPT1, AtIPT3, AtIPT5, and AtIPT8) were found in plastids, in agreement with the idea that the MEP pathway primarily provides DMAPP to tZ and isopentenyladenine. On the other hand, AtIPT2, a tRNA isopentenyltransferase, was detected in the cytosol. Because the prenylated adenine moiety of tRNA is usually of the cZ type, the formation of cZ in Arabidopsis seedlings might involve the transfer of DMAPP from the MVA pathway to tRNA. Distinct origins of large proportions of DMAPP for tZ and cZ biosynthesis suggest that plants are able to separately modulate the level of these cytokinin species.