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J. Biol. Chem., Vol. 279, Issue 14, 14049-14054, April 2, 2004

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Distinct Isoprenoid Origins of cis- and trans-Zeatin Biosyntheses in Arabidopsis^*

Hiroyuki Kasahara, Kentaro Takei¶, Nanae Ueda¶, Shojiro Hishiyama||, Tomoyuki Yamaya¶, Yuji Kamiya, Shinjiro Yamaguchi, and Hitoshi Sakakibara¶**

From the Laboratory for Cellular Growth and Development and the ^¶Laboratory for Communication Mechanisms, Plant Science Center, RIKEN (The Institute of Physical and Chemical Research), Yokohama 230-0045, Japan and the ^||Forestry and Forest Products Research Institute, Ibaraki 305-8687, Japan

Received for publication, December 26, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 ¹³C in Arabidopsis seedlings. Efficient ¹³C labeling was achieved by blocking the endogenous pathway genetically or chemically during the feed of a ¹³C 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cytokinins (CKs),¹ a group of phytohormones, have profound physiological roles in plants, e.g. promotion of cell division, release of lateral buds from apical dominance, and delay of senescence. The biological activity, signal transduction, and metabolism of CKs have long been studied (1). However, it remains unclear how different classes of CKs are produced in plants and whether such classes of CKs play different roles in plant development. Most natural CKs are derivatives of N⁶-prenylated adenine. At least two CK species, trans-zeatin (tZ) and isopentenyladenine (iP), are considered to be active forms in Arabidopsis according to the specific recognition by a CK receptor that has recently been identified (2, 3). By contrast, cis-zeatin (cZ) exhibits only low or no activity to this Arabidopsis receptor (2).

In CK biosynthesis, the prenylation of AMP is catalyzed by adenylate isopentenyltransferase (adenylate-IPT; EC 2.5.1.27 [EC] ), which utilizes dimethylallyl diphosphate (DMAPP) as a substrate. This enzyme activity, leading to the formation of isopentenyladenine riboside monophosphate (iPRMP), was first demonstrated in a slime mold, Dictyostelium discoideum (4). Thereafter, a CK biosynthesis gene, tmr, which encodes adenylate-IPT, was isolated from a crown gall-inducing bacterium, Agrobacterium tumefaciens (5, 6). Recently, the enzymatic properties of Arabidopsis adenylate-IPT-like proteins (AtIPT1, AtIPT3–AtIPT8) have been determined (7, 8). Unlike tmr, some AtIPTs preferred ADP and ATP, rather than AMP, as a substrate to produce corresponding nucleotide CKs in vitro (8, 9). Hence, we refer to these enzymes as adenosine phosphate-IPTs. Subsequent conversion of iPRMP to iP is catalyzed by 5'-nucleotidase and adenosine nucleosidase (10, 11). The conversion of iP into tZ is catalyzed by microsomal trans-hydroxylase, which is probably a P450 monooxygenase (12).

On the other hand, the tRNA-dependent CK biosynthesis pathway has also been proposed in plants, as some tRNA species contain an N⁶-prenylated adenine moiety, which, by hydrolysis, is capable of forming CKs (13). The prenylation of tRNA is catalyzed by tRNA isopentenyltransferase (tRNA-IPT; EC 2.5.1.8 [EC] ). In Arabidopsis, AtIPT2 encodes a tRNA-IPT (14). AtIPT9 is also similar to bacterial tRNA-IPT in sequence (8), though its enzyme activity has not been demonstrated. In plants, cZ and iP are generally the major components of prenylated tRNAs (13). Therefore, the prenylated tRNA has been considered as a possible source of cZ. In addition, the occurrence of zeatin cis-trans isomerase activity (15) suggests that the tRNA-mediated pathway might also contribute to the synthesis of tZ-type CKs through cZ-type CKs.

Plants have two possible biosynthesis pathways for the prenyl group of CKs, the methylerythritol phosphate (MEP) pathway in plastids and the mevalonate (MVA) pathway in the cytosol (16, 17). Both pathways supply the common isoprenoid precursors isopentenyl diphosphate and DMAPP. Although the MEP and MVA pathways exist in separate subcellular locations, there is some exchange of common precursor(s) between the two pathways (18, 19). Therefore, administration of a MEP pathway precursor can partially suppress the growth inhibition caused by a block in the MVA pathway and vice versa (20). As is the case with other isoprenoid biosyntheses, the MVA pathway had been considered the sole route for providing DMAPP to CKs until the MEP pathway was uncovered recently. In fact, the incorporation of ¹⁴C labeled MVA into the iP element of tRNA in vivo has been demonstrated in tobacco pith tissue (21). The incorporation of [¹³C]MVA into iP and trans-zeatin riboside (tZR) in vitro was also reported in the endosperm of Sechium edule seeds (22). In addition, there are some reports that indicate that CK levels are reduced in plants when the MVA pathway is limited (23–25). On the other hand, the contribution of the MEP pathway to CK biosynthesis has never been studied previously. It should be noted that the incorporation of MVA does not exclude a potential role of the MEP pathway in the biosynthesis of CKs; it has been observed that isoprene units from both MEP and MVA pathways are incorporated into a single downstream isoprenoid (19, 20).

To selectively label metabolites from the MEP and MVA pathways with ¹³C in vivo, we have previously performed feeding of ¹³C labeled 1-deoxy-D-xylulose (DX) or mevalonolactone (MVL) to Arabidopsis seedlings (19). DX is converted into the MEP pathway intermediate 1-deoxy-D-xylulose 5-phosphate by phosphorylation. Therefore, exogenous DX is able to complement the albino phenotype of the cla1-1 mutant (26), which is defective in 1-deoxy-D-xylulose-5-phosphate synthase in the MEP pathway. Similarly, the growth inhibition due to a block in the MVA pathway by mevastatin (an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase) is rescued by exogenous application of MVL (19). Efficient ¹³C labeling of metabolites from the MEP and MVA pathways was thus achieved by feeding ¹³C labeled DX and MVL to the cla1-1 mutant and mevastatin-treated plants, respectively, at a concentration that is sufficient to restore the phenotype nearly fully to wild type. This method allowed us to determine the contribution of the MEP and MVA pathways to the biosynthesis of gibberellins, another group of phytohormones, by gas chromatography-mass spectrometry (GC-MS) (19).

In this study, we address the biosynthesis route for the prenyl moiety of CKs using the ¹³C labeled tracers in Arabidopsis seedlings. Our data demonstrate that the prenyl side chains of tZ- and iP-type CKs are mainly produced through the MEP pathway, whereas a large fraction of cZ derivatives is synthesized through the MVA pathway. We also show the subcellular location of AtIPTs produced as green fluorescent protein (GFP)-fusion proteins. Based on these data, we propose a crucial role of the plastid-localized MEP pathway in CK biosynthesis, and discuss how different classes of CKs are biosynthesized through the MEP and MVA pathways in plants.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Plant Materials, Growth Conditions, and Chemicals—Arabidopsis thaliana ecotype Wassilewskija-2 was used for ¹³C labeling experiments. Feeding of [1-¹³C]DX and [2-¹³C]MVL was carried out in the presence of 1% sucrose as described previously (19), except that uniconazole treatment was omitted. A. thaliana ecotype Columbia-0 was used for observation of GFP fluorescence. DX and [1-¹³C]DX (99% labeled) were synthesized as reported before using iodomethane and [¹³C]-iodomethane (99% labeled, Aldrich), respectively (27). MVL (DL-mevalonolactone) and [2-¹³C]MVL (99% labeled) were purchased from Aldrich, and mevastatin was purchased from Calbiochem.

Particle Bombardment—Full-length or part of the coding regions of AtIPTs (AtIPT1, Met¹–Leu⁷¹; AtIPT2, Met¹–Asn⁴⁶⁶; AtIPT3, Met¹–Ser⁵⁵; AtIPT4, Met¹–Asn³¹⁸; AtIPT5, Met¹–Ser⁴⁷; AtIPT7, Met¹–Phe²⁹; AtIPT8, Met¹–Val³³⁰) were fused to the amino terminus of the GFP gene, which was controlled by the cauliflower mosaic virus 35 S promoter (35S-sGFP (S65T)) (28). The DNA constructs were introduced into the roots or rosette leaves of 2- or 3-week-old seedlings by particle bombardment (PDU-1000/He, Bio-Rad). Transient expression was observed by laser confocal-scanning fluorescence microscopy after overnight incubation (Fluoview IX5, Olympus).

Stable Transformants of Arabidopsis—Genomic DNA fragments of AtIPT3 and AtIPT7 containing the 5'-flanking regions (3.9 kb for AtIPT3, 4.1 kb for AtIPT7) and the full coding regions were ligated into pTH2 vectors (29) to produce chimeric genes fused to the amino terminus of the GFP gene. The chimeric genes were introduced into Arabidopsis by the floral dip method (30). More than five independent lines were obtained, and T3 plants were used for analysis.

Plant Hormone Analysis—Extraction and fractionation of CKs from Arabidopsis seedlings (3–4 g) were performed as described previously (31). The nucleotide CK fractions were further analyzed as the corresponding nucleosides after treatment with phosphatase (9). Each CK fraction was purified by immunoaffinity columns (32) except for the addition of anti-cis-zeatin riboside (cZR) antibodies to the original protocol. After desalting, the resulting samples were dissolved in H₂O and analyzed with a liquid chromatography (LC)-MS system (model 2695/ZQ2000MS, Waters). CKs were separated at a flow rate of 0.25 ml/min, with the gradients of solvents A (H₂O), B (methanol), and C (0.1% acetic acid) set according to the following profile: 0 min, 95% A + 5% C; 1 min, 95% A + 5% C; 16 min, 45% A + 50% B + 5% C; 22 min, 25% A + 70% B + 5% C. Capillary voltage was 4.0 kV. Other conditions were described previously (9). The quantification of [M+H]⁺ and +1 signals were achieved at levels between 0.1 and 100 pmol/injection using a standard curve. Abscisic acid (ABA) was purified from the methanoleluted fraction from the MCX column (Waters) in CK purification by high performance liquid chromatography and analyzed by GC-MS as reported previously (33). The ¹³C incorporation level was calculated using the fragment ion cluster between m/z 190 and 192 after subtraction of natural ¹³C abundance (34). Campesterol (CAM) was eluted from the MCX column with CH₂Cl₂ (3 ml x 3), after purification of CKs. CAM was further purified by high pressure liquid chromatography and analyzed by GC-MS as reported previously (19).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
CK Levels in Arabidopsis Seedlings—To study the metabolic origins of the prenyl group of CKs, we planned to conduct the ¹³C labeling experiment that was employed previously to determine the contribution of the MEP and MVA pathways to gibberellin biosynthesis (19). To examine whether this method was feasible for CKs, we first analyzed endogenous CK levels by LC-MS in Arabidopsis seedlings under the same growth conditions, after a feeding of non-labeled DX and MVL. When DX was fed to the cla1-1 mutant to rescue its albino phenotype, all CKs analyzed were detectable by LC-MS. As shown in Table I, the trend in the amount of individual CKs is as follows: nucleotide CKs > nucleoside CK > free-base CKs (e.g. trans-zeatin riboside monophosphate (tZRMP) > tZR > tZ). It was also noted that cZ-type CKs accumulated at much lower levels relative to tZ- and iP-type CKs. Similar accumulation patterns of different CK species were observed when the growth inhibition by mevastatin was rescued by MVL (Table I). These data allowed us to confirm that for all CKs examined, an accurate quantification of the molecular ion and its +1 isotopomer ion by LC-MS would be possible in this system with the exception of cZ, which was detectable, but its +1 isotopomer ion was below the range of reliable quantification.

View larger version (14K): [in this window] [in a new window]   FIG. 1. Predicted 13C labeling patterns for CKs in [1-13C]DX-fed cla1-1 seedlings and [2-13C]MVL-fed wild type seedlings. Red arrows indicate the possible metabolic conversions of [1-13C]DX through the MEP pathway and blue arrows are the possible metabolic conversions of [2-13C]MVL through the MVA pathway. Asterisks show the position of the 13C label. A possible metabolic conversion of [1-13C]DX to cZ-type CKs through the MEP pathway and a possible metabolic conversion of [2-13C]MVL to tZ-type CKs through the MVA pathway are not shown. Dashed blue arrows indicate that the cZ biosynthesis through iP-type CKs is fairly hypothetical. P and PP represent monophospholic and diphospholic acids, respectively. GAP, glyceraldehyde 3-phosphate; PYR, pyruvate.

View larger version (15K): [in this window] [in a new window]   FIG. 2. Relative levels of 13C incorporation into CKs, ABA, and CAM in [1-13C]DX-fed Arabidopsis cla1-1 seedlings (A) and in [2-13C]MVL-fed wild type seedlings in the presence of mevastatin (B). The 13C incorporation level was calculated using the molecular ion cluster shown in Table II after subtraction of natural 13C abundance. 13C incorporation levels were measured in three independent experiments. Error bars indicate S.D.

Subcellular Localization of AtIPTs—Because the prenyl group of tZ- and iP-type CKs is predominantly synthesized through the MEP pathway (Fig. 2), we speculated that some IPTs should use the DMAPP produced in plastids. Compared with the bacterial enzyme tmr, all AtIPTs except for AtIPT4 have an amino-terminal extension consisting of 30–60 amino acids (7). The ChloroP 1.1 program (36) predicted that the amino-terminal regions of AtIPT1, -3, -5, and -8 might function to localize these AtIPTs to plastids. To obtain experimental evidence for the subcellular locations of IPT activities, we prepared DNA constructs designed to produce translational fusions of individual AtIPTs and GFP. Because some of the fusion proteins containing the full coding regions of AtIPTs tended to precipitate in cells (data not shown), the amino-terminal regions, which could function as transit peptides, were fused to GFP (for details, see "Experimental Procedures"). The plasmid DNAs were introduced into both Arabidopsis leaf and root cells, and the transient expression of the chimeric genes was observed. The fluorescence of AtIPT1-, AtIPT3-, and AtIPT5-GFP in mesophyll cells was observed in plastids as the signal overlapped with autofluorescence derived from chlorophylls (Fig. 3, A, B, D, and G). AtIPT8-GFP exhibited the same fluorescence pattern as AtIPT1 and -3 in root cells (Fig. 3, C, E, and H). However, the fluorescence of AtIPT4- and AtIPT2-GFP was distributed in the cytosol (Fig. 3, I, M, and N). It should be noted that an identical pattern of GFP fluorescence was observed when the GFP was fused to the carboxyl terminus of AtIPT4 (data not shown). The fluorescence of AtIPT7-GFP was observed in the mitochondria (Fig. 3, J and K). To avoid mislocation of the fusion proteins because of transient expression by a strong promoter, we next generated stable transformants of Arabidopsis expressing AtIPT3- and AtIPT7-GFP controlled by their native promoters. Consequently, the pattern of fluorescence produced by both of these constructs was essentially the same as the respective transiently expressed fusion proteins (Fig. 3, compare E with F and K with L). These results strongly suggest that four adenosine phosphate-IPTs, AtIPT1, -3, -5, and -8, localize in plastids in agreement with the idea that the MEP pathway primarily provides DMAPP for tZ and iP synthesis. As for tRNA-IPT, AtIPT2-GFP localized in the cytosol (Fig. 3, M and N).

View larger version (39K): [in this window] [in a new window]   FIG. 3. Subcellular localization of AtIPT-GFP fusion proteins in Arabidopsis. The fusion constructs expressing AtIPT1-GFP (A–C), AtIPT3-GFP (D and E), AtIPT5-GFP (G), AtIPT8-GFP (H), AtIPT4-GFP (I), AtIPT7-GFP (J and K), and AtIPT2-GFP (M and N) were introduced into leaf cells (A, B, D, G, J, M, N) and root cells (C, E, H, I, K) by particle bombardment. Stable transformants expressing AtIPT3-GFP (F) and AtIPT7-GFP (L) driven by their own promoters were generated. Fluorescence was observed using a confocal laser-scanning microscope. Chlorophyll autofluorescence was merged on the GFP fluorescence images in B, D, G, J, and N. Transmission images were superimposed on the fluorescence images in C, E, F, H, K, and L. Bar = 10 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Using a ¹³C labeled precursor specific to the MEP and MVA pathways, we have demonstrated that both isoprenoid pathways can supply DMAPP to CKs in Arabidopsis seedlings (Fig. 2). In addition, subcellular localization of AtIPT-GFP fusion proteins has been determined in Arabidopsis cells. The results from GFP fluorescence observations suggest that IPT isozymes are distributed to multiple subcellular compartments (Fig. 4). These data now provide new insights into CK biosynthesis as discussed below.

View larger version (65K): [in this window] [in a new window]   FIG. 4. Proposed CK biosynthesis pathway and the localization of related enzymes in Arabidopsis. Red arrows indicate possible biosynthesis pathways for iP and tZ through the MEP pathway, and blue arrows are those proposed for cZ through the MVA pathway. Black arrows depict possible cis-trans isomerization between tZ- and cZ-type CKs. The non-filled red and blue arrows indicate a hypothetical exchange of common precursor(s) between the MEP and MVA pathways. The non-filled black arrow indicates a predicted contribution of the MVA pathway to mitochondrial isoprenoid biosynthesis. The dashed arrows denote multiple steps. GAP, glyceraldehyde 3-phosphate; PYR, pyruvate.

There are at least two possible explanations for the minor incorporation of [2-¹³C]MVL into tZ- and iP-type CKs (Fig. 2). First, as has been suggested previously, there may be some exchange of common isoprenoid precursors (e.g. isopentenyl diphosphate or DMAPP) between the cytosol and plastids. This mechanism has been proposed to explain the minor incorporation of [2-¹³C]MVL into ent-kaurene (a precursor for gibberellins), assuming that ent-kaurene synthesis occurs exclusively in plastids as supported by the subcellular localization of enzymes (38). However, in the case of the biosynthesis of tZ- and iP-type CKs, a small amount of ¹³C label from [2-¹³C]MVL can also be attributed to the function of another adenosine phosphate-IPT, AtIPT4, which is presumably present in the cytosol (Fig. 3I). In addition, the localization of AtIPT7-GFP in the mitochondria (Fig. 3, J–L) suggests that this enzyme may also use DMAPP from the MVA pathway, because ubiquinones, a group of isoprenoids in mitochondria, are synthesized principally through the MVA pathway (39). Therefore, a major role of the MEP pathway in the biosynthesis of tZ- and iP-type CK in Arabidopsis seedlings under the current growth conditions does not rule out a greater contribution of the MVA pathway to these CKs under different growth conditions, if the relative abundance of IPT isozymes is modulated. Thus, our present results do not contradict previous reports (22–24) indicating a role for the MVA pathway in providing DMAPP for tZ-type CKs. In this context, to fully understand the relative roles of the MEP and MVA pathways in CK biosynthesis, it will be informative to determine how individual AtIPT genes are regulated during plant development and under different environmental conditions.

Recently, Åstot et al. (24) measured the biosynthetic rates of tZRMP and iPRMP using in vivo isotope labeling and proposed an iPRMP-independent pathway for the biosynthesis of tZ-type CKs. In this model, a hydroxylated derivative of DMAPP is directly transferred to AMP (24). In light of this finding, our result showing the involvement of the MEP pathway in tZ biosynthesis is intriguing because hydroxymethylbutenyl diphosphate, the best hypothetical substrate in the iPRMP-independent pathway (24), has been known as an intermediate of the MEP pathway (Fig. 4) (40). In fact, it has been demonstrated recently (41) that TZS, an IPT from Agrobacterium, is capable of synthesizing tZRMP from hydroxymethylbutenyl diphosphate and AMP in vitro. To examine whether the iPRMP-independent pathway operates in plants, hydroxymethylbutenyl diphosphate should be tested as a substrate for AtIPT1, -3, -5, and -8 in vitro.

The Origin of DMAPP for cZ-type CKs—Based on the efficient ¹³C incorporation of [2-¹³C]MVL into cZ derivatives, relative to that into ABA and other CKs (Fig. 2), we postulate that a large portion of the prenyl group of cZ-type CKs originates from the MVA pathway. However, the level of [1-¹³C]DX incorporation into cZ-type CKs was also significantly higher than incorporation into CAM, a cytosolic sterol that is mainly produced through the MVA pathway. Therefore, the MEP pathway appears to supply DMAPP to cZ derivatives at a level beyond the hypothesized exchange of isoprenoid precursors (Fig. 4). Unlike the possible participation of a P450 monooxygenase in the hydroxylation of iP to form tZ (12), the conversion of iP-type CKs into their corresponding cZ derivatives has not been reported. Therefore, this route cannot readily be hypothesized at the moment.

A probable explanation for the incorporation of MEP-derived DMAPP into cZ derivatives is isomerization of tZ-type CKs (Fig. 4). The presence of a cis-trans isomerase of zeatin has been reported in cell-free extracts from immature seeds of Phaseolus vulgaris (15). This enzyme catalyzes the isomerization of zeatin in both directions in favor of cZ to tZ and uses cZR and tZR as substrates as well. In our experimental conditions, the amounts of cZ-type CKs were much smaller than those of corresponding tZ-type CKs (Table I), and the majority of tZ- type CKs were labeled with ¹³C in cla1-1 seedlings fed with [1-¹³C]DX (Fig. 2). Thus, even a small proportion of isomerization of tZ to cZ would account for the increase in ¹³C incorporation from [1-¹³C]DX into cZ-type CKs. In contrast, the rate of ¹³C incorporation into tZ-type CKs from [1-¹³C]DX would not be greatly affected by isomerization of cZ-type CKs because of their low levels in the current experimental system.

A role of the tRNA-dependent pathway has been proposed in the biosynthesis of cZ-type CKs, because the N⁶-prenylated adenine element of tRNA generally consists of iP- and cZ-type CKs but not tZ-type CKs (13). In addition, no hydroxylase activity has ever been shown to produce cZ derivatives from iP-type CKs. Our finding that the AtIPT2-GFP fusion protein localizes to the cytosol (Fig. 3) suggests that the N⁶-prenylation of tRNA takes place in this subcellular compartment in Arabidopsis and implies the use of DMAPP derived from the MVA pathway. In fact, our ¹³C labeling experiments indicated that a large proportion of cZ derivatives are synthesized through the MVA pathway (Fig. 2), supporting the role of N⁶-prenylated tRNA in the biosynthesis of cZ-type CKs (Fig. 4). The incorporation of ¹⁴C labeled MVA into the iP element of tRNA has been demonstrated previously in tobacco pith tissue (21).

In conclusion, the present study has demonstrated a critical contribution of the plastid-located MEP pathway in providing the prenyl precursor to tZ- and iP-type CKs in Arabidopsis seedlings. This conclusion is well supported by the localization of multiple AtIPT-GFP fusion proteins in plastids. In contrast, it is likely that the MVA pathway is mainly responsible for the biosynthesis of cZ derivatives, although our data do not exclude the possibility that a small fraction of cZ derivatives may be produced through isomerization of tZ-type CKs. It is still not clear whether cZ plays any critical role in plant development; however, the distinct origins of large fractions of DMAPP for tZ and cZ biosynthesis suggest that plants are able to separately modulate the level of these CK species. Further characterization of the biosynthesis pathway for cZ would be necessary to evaluate its physiological significance in plants. Additional work is required to uncover how multiple IPTs in varying subcellular compartments control the levels of different classes of CKs during plant development and in response to environmental cues.

	FOOTNOTES

 
^* This study was supported in part by Grants-in-aid for scientific research (12142202 (to H. S.) and 15770035 (to K. T.)) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^** To whom correspondence should be addressed: Plant Science Center, RIKEN, Suehiro 1-7-22, Tsurumi, Yokohama 230-0045, Japan. Tel.: 81-45-503-9576; Fax: 81-45-503-9609; E-mail: sakaki{at}postman.riken.go.jp.

¹ The abbreviations used are: CK, cytokinin; ABA, abscisic acid; CAM, campesterol; cZ, cis-zeatin; cZR, cis-zeatin riboside; cZRMP, cis-zeatin riboside monophosphate; DMAPP, dimethylallyl diphosphate; DX, 1-deoxy-D-xylulose; GFP, green fluorescent protein; iP, isopentenyladenine; iPR, isopentenyladenine riboside; iPRMP, isopentenyladenine riboside monophosphate; IPT, isopentenyltransferase; GC-MS, gas chromatography-mass spectrometry; LC-MS, liquid chromatography-MS; MEP, methylerythritol phosphate; MVA, mevalonate; MVL, mevalonolactone; tZ, trans-zeatin; tZR, trans-zeatin riboside; tZRMP, trans-zeatin riboside monophosphate.

	ACKNOWLEDGMENTS

 
We thank Drs. Damian O'Neill and Eiji Nambara (RIKEN) and Tomohisa Kuzuyama (The University of Tokyo, Tokyo) for helpful comments on the manuscript. We also thank Masanori Okamoto (Tokyo Metropolitan University, Tokyo) for help on the GC-MS analysis of ABA.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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