Plant Cell Physiol. 42(7): 677–685 (2001)
JSPP © 2001
Identification of Plant Cytokinin Biosynthetic Enzymes as Dimethylallyl
Diphosphate:ATP/ADP Isopentenyltransferases
Tatsuo Kakimoto 1
Department of Biology, Graduate School of Science, Osaka University, and Recognition and Formation, PRESTO, JST, Osaka, 560-0043 Japan
;
presence of auxins, and induce shoot formation on calli (Skoog
and Miller 1957). They also release axillary buds from apical
dominance, increase sink strength, and delay senescence (Mok
1994).
Two biosynthetic pathways of cytokinins, the tRNA pathway and the AMP pathway, have been proposed. In the first
pathway, tRNA has been speculated to be a possible source of
cytokinins, because an adenine residue in a subset of tRNAs is
isopentenylated. However, this pathway is not considered to be
the main route, if at all (Chen 1997, McGaw and Burch 1995).
In the AMP pathway, the isopentenyl group is transferred from
dimethylallyl diphosphate (DMAPP) to the N6 of AMP, resulting in the production of isopentenyladenosine-5¢-monophosphate (iPMP). This reaction is thought to be catalyzed by
DMAPP:AMP isopentenyltransferases. Several gall-forming
phytopathogenic bacteria possess genes for this class of
enzymes, e.g., tzs and ipt (same as tmr) of Agrobacterium
tumefaciens (Barry et al. 1984, Akiyoshi et al. 1984, Morris et
al. 1993), ptz of Pseudomonas savastanoi (Powell and Morris
1986), ipt of Rhodococcus fasciens (Crespi et al. 1992), and ipt
(same as etz) of Erwinia harbicola (Lichter et al. 1995). The
expression of tzs in A. tumefaciens results in secretion of cytokinins, whereas ipt in A. tumefaciens is integrated into plant
genomes to force the host plants to produce cytokinins and
form tumors. This activity has also been found in crude
extracts from cytokinin-autotrophic tobacco callus (Chen and
Melitz 1979) and from immature maize kernels (Blackwell and
Horgan 1994), but the corresponding enzymes have not been
purified to homogeneity. Recently, the presence of another
pathway was demonstrated in Arabidopsis thaliana, although
the reactions that constitute this pathway have not been identified yet. In this pathway, trans-zeartin-riboside monophosphate (ZMP) is synthesized independently of iPMP (Åstot et al.
2000). In the present study, a search of Arabidopsis genome
sequences for proteins that could code for isopentenyltransferases resulted in the identification of nine putative genes for
isopentenyltransferases. The same set of genes were also identified by Takei et al. (2001), who demonstrated that crude
extracts of E. coli expressing these genes and purified AtIPT1
had DMAPP:AMP isopentenyltransferase activity. Here I
report that one of these genes, AtIPT4, has unique
DMAPP:ATP/ADP isopentenyltransferase activity in its puri-
It has been believed that the key step in cytokinin biosynthesis is the addition of a 5-carbon chain to the N6 of
AMP. To identify cytokinin biosynthesis enzymes that catalyze the formation of the isopentenyl side chain of cytokinins, the Arabidopsis genomic sequence was searched for
genes that could code for isopentenyltransferases. This
resulted in the identification of nine putative genes for isopentenyltransferases. One of these, AtIPT4, was subjected
to detailed analysis. Overexpression of AtIPT4 caused cytokinin-independent shoot formation on calli. As shoot formation on calli normally occurs only when cytokinins are
applied, it suggested that this gene product catalyzed cytokinin biosynthesis in plants. Recombinant AtIPT4 catalyzed the transfer of an isopentenyl group from dimethylallyl diphosphate to the N6 of ATP and ADP, but not to that
of AMP. AtIPT4 did not exhibit the DMAPP:tRNA isopentenyltransferase activity. These results indicate that cytokinins are, at least in part, synthesized from ATP and ADP in
plants.
Key words: Adenosine diphosphate — Adenosine triphosphate — Arabidopsis thaliana — Cytokinin — Dimethylallyl
diphosphate:ATP/ADP isopentenyltransferase.
Abbreviations: DMAPP, dimethylallyl diphosphate; CIAP, calf
intestine alkaline phosphatase; iPA, isopentenyladenosine; iPDP, isopentenyladenosine-5¢-diphosphate; iPMP, isopentenyladenosine-5¢monophosphate; IPTG, isopropyl-b-D-thiogalactopyranoside; iPTP, isopentenyladenosine-5¢-triphosphate; RT, reverse transcription; WS,
Wassilewskija; ZDP, trans-zeatin-riboside diphosphate; ZMP, transzeatin-riboside monophosphate; ZTP, trans-zeatin-riboside triphosphate.
The nucleotide sequences of AtIPT1, AtIPT3, AtIPT4, AtIPT5,
AtIPT6, AtIPT7 and AtIPT8 of Arabidopsis (strain, WS) have been
submitted to DDBJ under accession numbers of AB061400,
AB061401, AB061402, AB061403, AB061404, AB061405 and
AB061406, respectively.
Introduction
Cytokinins are a class of plant hormones that play a pivotal role in plant development. They induce cell division in the
1
Corresponding author: E-mail, [email protected]; Fax, +81-6-6850-5420; Phone, +81-6-6850-5421.
677
678
New cytokinin biosynthetic route
fied form, and is functional in plants. We propose that one of
the key steps, or perhaps the key step, in cytokinin biosynthesis is the isopentenylation of ATP and/or ADP.
TACTCCATCTCATCTACGGA-3¢) and No. 487 (5¢-CGGGATCCTCACCGAATTCGCGTCAGCGT-3¢). The amplified fragment was
digested with NdeI and BamHI, and cloned between the NdeI and
BamHI sites of pER16b. All regions produced by PCR were confirmed by sequencing.
Materials and Methods
Identification of genes for isopentenyltransferases
A reverse transcription-polymerase chain reaction (RT-PCR) was
used to confirm the expression and isolation of cDNA sequences.
DNase-treated RNA samples from various organs of A. thaliana var.
Wassilewskija (WS) were used as templates. Primers were designed to
amplify regions encompassing the entire coding regions for putative
isopentenyltransferases, and the amplified regions were sequenced.
Plasmid construction
The coding region of AtIPT4, which has no intron, was PCRamplified from genomic DNA of Arabidopsis (WS strain) by using
primers No. 421 (5¢-AAAATGAAGTGTAATGACAAAATGGTTGTG3¢) and No. 407 (5¢-GTCCAAACTAGTTAAGACTTAAAAATC-3¢).
The amplified fragment was blunt-end cloned under the cauliflower
mosaic virus 35S promoter of a plant expression binary vector,
pTK015 (Kakimoto unpublished), resulting in 35S::AtIPT4. The coding region of AtIPT2 was amplified from an Arabidopsis (WS) cDNA
library by using primers No. 398 (5¢-TCCCCCGGGCGATGATGATGTTAAACCCTAGC-3¢) and No. 399 (5¢-TCCCCCGGGTCAATTTACTTCTGCTTCTTGAACTTC-3¢). The amplified fragment was
digested with SmaI and cloned under the 35S promoter of pTK015,
resulting in 35S::AtIPT2. A DNA fragment corresponding to the coding region of AtIPT4, amplified with the use of primers No. 480 (5¢GGAATTCCATATGAAGTGTAATGACAAAATGGTTG-3¢) and No.
481 (5¢-GAAGATCTGTCCAAACTAGTTAAGACTTAAAAATC-3¢),
was digested with NdeI and BglII and cloned between the NdeI and
BamHI sites of pET16b (Novagen, Madison, WI, U.S.A.), which was
used to express AtIPT4 as a polyhistidine fusion protein in E. coli. The
coding region of AtIPT4 was also amplified with the use of primers
No. 483 (5¢-AAGTGTAATGACAAAATGGTTGTGATCATG-3¢) and
No. 481, and the amplified fragment was digested with NcoI and BglII
and cloned between the NcoI and BamHI sites of pET32b (Novagen),
producing pET32-AtIPT4. The coding region of AtIPT2 was amplified from 35S::AtIPT2 with the use of primers No.550 (5¢-GATCCCCGGCATATGATGATGTTAAACCCTAGC-3¢) and No.551 (5¢ACGGTACCCATATGTCAATTTACTTCTGCTTCTTGAAC-3¢), and
the amplified fragment was digested with NdeI and cloned into the
NdeI site of pET16b. The miaA gene of E. coli was PCR-amplified
from whole E. coli cells (DH10B) with the use of primers No. 485
(5¢-GAAGATCTTCAGCCTGCGATAGCACCAACAA-3¢) and No.
484 (5¢-GGAATTCCATATGAGTGATATCAGTAAGGCGAG-3¢). The
amplified fragment was digested with NdeI and BglII, and then cloned
between the NdeI and BamHI sites of pET16b. The tzs gene of Agrobacterium was PCR-amplified from whole A. tumefaciens cells
(GV3101) with the use of primers No. 486 (5¢-GGAATTCCATATGT-
Transformation of calli
Calli of Arabidopsis were transformed according to the method
of Kakimoto (1998). Selection for transformants was made at 50 mg
ml–1 kanamycin sulfate.
Expression and purification of recombinant proteins
The E. coli strain AD494(DE3)pLysS (Novagen), harboring an
expression plasmid, was cultured overnight at 20°C in the presence of
1 mM isopropyl-b-D-thiogalactopyranoside (IPTG), harvested, and
resuspended in buffer [25 mM Tris·HCl, pH 7.5; 50 mM KCl; 5 mM
2-mercaptoethanol; 0.5 mM phenylmethylsulfonyl fluoride (PMSF);
10 mg ml–1 leupeptin] at a cell density of 100 OD600. This E. coli suspension was frozen and thawed to disrupt the cells, and then centrifuged at 300,000´g for 10 min, and the supernatant was collected.
Supernatants (referred to as crude extracts in this paper) were either
used for assay of isopentenyltransferase activity, or subjected to affinity purification of enzymes by Ni-NTA agarose (Qiagen, Tokyo,
Japan). Ni-NTA agarose was suspended in 4´ binding buffer (20 mM
Na-phosphate buffer, pH 8.0; 1.2 M NaCl; 40 mM imidazole, pH 8.0;
5 mM 2-mercaptoethanol) at a 50% bed volume. This suspension was
mixed with three volumes of E. coli extracts, and incubated at 4°C for
1 h with gentle mixing. It was then centrifuged to separate the supernatant (unbound fractions) from the Ni-NTA agarose pellets. The NiNTA agarose pellets were washed thoroughly with wash buffer
(20 mM Na-phosphate, pH 8.0; 10 mM imidazole, pH 8.0; 0.3 M
NaCl; 5 mM 2-mercaptoethanol; 10 mg ml–1 leupeptin; 0.3 mM
PMSF). Recombinant proteins were eluted with elution buffer (20 mM
Na-phosphate buffer, pH 8.0; 0.3 M imidazole, pH 8.0; 0.3 M NaCl;
5 mM 2-mercaptoethanol; 10 mg ml–1 leupeptin), and concentrated by
using the ultrafiltration membrane UFV5BCC25 (Millipore Co., Bedford, MA, U.S.A.), with replacing the elution buffer by another buffer
(25 mM Tris·HCl, pH7.5; 50 mM KCl; 5 mM 2-mercaptoethanol;
10 mg ml–1 leupeptin). Recombinant proteins were stored in liquid
nitrogen.
Assay for DMAPP:ATP (or ADP or AMP) isopentenyltransferase
activity
DMAPP:ATP (or ADP or AMP) isopentenyltransferase activity
was measured by the method described by Blackwell and Horgan
(1991), with some modifications. The samples to be assayed were
crude extracts and purified proteins of E. coli harboring the pET16bderivatives. Purified proteins were diluted to the concentrations
indicated in Table 1 and Table 2 with dilution buffer (25 mM Tris·HCl,
pH 7.5; 5 mM 2-mercaptoethanol; 0.2 mg ml–1 bovine serum albumin).
Isopentenylation reactions were started by mixing samples with an
equal volume of 2´ assay mixture containing 25 mM Tris·HCl (pH 7.5),
Fig. 1 (A) Alignment of a conserved region of isopentenyltransferases. The horizontal bar indicates an ATP/GTP binding motif. Numbers in
parentheses indicate the numbers of amino acid residues present before and after the conserved region of each gene product. Identical amino acid
residues conserved in more than 50% of the gene products are shaded with dark gray, and similar amino acid residues conserved in more than
50% of the gene products are shaded with light gray. (B) A phylogenetic tree for the conserved region of isopentenyltransferases. The tree was
made with the ClustalW program (http://www.ddbj.nig.ac.jp/E-mail/clustalw-e.html), with bootstrapping. Bar, 0.1 amino acid substitutions per
site. For (A) and (B), the gene products are colored as described below. Plant gene products are shown in pink (putative cytokinin biosynthetic
isopentenyltransferases) or in orange (putative DMAPP:tRNA isopentenyltransferases). Non-plant eukaryotic DMAPP:tRNA isopentenyltransferases are dark blue, and bacterial DMAPP:tRNA isopentenyltransferases are light blue. Cytokinin biosynthetic isopentenyltransferases of gallforming phytopathogenic bacteria are green.
New cytokinin biosynthetic route
10 mM MgCl2, 5 mM 2-mercaptoethanol, 60 mM DMAPP, and 2 mM
[2,8-3H]ATP (120 GBq mmol–1), [2,8-3H]ADP (118 GBq mmol–1), [23
H]AMP (72 GBq mmol–1), or [2,8-3H]adenosine (143 GBq mmol–1).
After incubation for the time indicated in the tables, 1/2 volume of calf
679
intestine alkaline phosphatase (CIAP) mix [0.5 M Tris·HCl (pH 9.0),
10 mM MgCl2, and 1,000 units ml–1 of CIAP (Takara Shuzo Co. Ltd.,
Otsu, Shiga, Japan)] was added and the mixtures were incubated at
37°C for 30 min. Then, 700 ml of ethyl acetate was added and the mix-
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New cytokinin biosynthetic route
tures were vortexed. After centrifugation at 17,000´g for 2 min, the
organic phase was recovered and washed twice with water. The
organic phase was mixed with ten volumes of scintillant, ACSII
(Amersham Pharmacia Biotech, Tokyo, Japan), and radioactivity
levels were measured with a liquid scintillation counter. Recovery of
[2,8-3H]isopentenyladenosine (iPA) was measured, and were used to
calculate the amounts of the products formed. The [2,8-3H]iPA had
been synthesized through isopentenylation of ATP by using purified
AtIPT4, followed by CIAP treatment as described above. All assays
were performed in duplicate, and mean values were used for calculation.
To determine the Km for ATP, purified protein (2 ng ml–1 in
dilution buffer) was mixed with the same volume of a 2´ assay
mixture containing 25 mM Tris·HCl (pH 7.5), 10 mM MgCl2, 5 mM 2mercaptoethanol, 0.4 mM DMAPP, and ATP (2–502 mM [2,8-3H]ATP,
1.22 MBq ml–1). To determine the Km for DMAPP, purified protein
(2 ng ml–1) was mixed with the same volume of a 2´ assay mixture
containing 25 mM Tris·HCl (pH 7.5), 10 mM MgCl2, 5 mM 2mercaptoethanol, 0.25–200 mM DMAPP, and 200 mM [2,8-3H]ATP
(7.07 GBq mmol–1). After the mixture had been incubated at 24°C for
0 min or 4 min, the reaction mixtures were treated with CIAP, and then
extracted with ethyl acetate as described above. Values obtained at
0 min were subtracted from those at 4 min, and the resulting differences were taken as enzyme activity.
Assay for DMAPP:tRNA isopentenyltransferase activity
Undermodified tRNA was prepared by permanganate-treatment
of yeast tRNA (typeX, Sigma-Aldrich Japan, Tokyo, Japan) according
to the method of Kline et al. (1969). Twenty microliters of purified
protein samples (20 ng (protein ml–1) in dilution buffer was mixed with
the same volume of 2´ tRNA isopentenyltransferase assay mixture
(25 mM Tris·HCl, pH 7.5; 10 mM MgCl2; 5 mM 2-mercaptoethanol;
0.67 mM [1-3H]DMAPP, 555 GBq mmol–1; and 567 A260 units ml–1
undermodified tRNA), and incubated at 25°C for 30 min. After 160 ml
of 0.4 M sodium acetate and 500 ml of ethanol was added and allowed
to settle on ice for 10 min, the tRNA precipitates were recovered by
centrifugation (17,000´g for 20 min), washed with 80% ethanol, and
dissolved in 30 ml of distilled water. These were mixed with ten volumes of ACSII, and radioactivity levels were measured. Assays were
performed in triplicate and mean values were used for calculation.
Analysis of product by HPLC and mass spectrometry
Crude extract prepared from IPTG-induced E. coli harboring
pET32-AtIPT4 was incubated with Ni-NTA agarose beads. After the
beads had been washed thoroughly, they were re-suspended in a solution containing 25 mM Tris·HCl (pH 7.5), 100 mM KCl, and 5 mM 2mercaptoethanol. The bead pellets were mixed with an equal volume
of a 2´ assay mixture that contained 1 mM unlabeled ATP and 1 mM
DMAPP, and incubated at 25°C for 1 h with shaking. After a brief
spin, the supernatant was recovered and separated into two portions,
and one portion was treated with CIAP as described before. The supernatant with or without treatment with CIAP was mixed with three volumes of acetone. The mixture was incubated at –80°C for 30 min and
centrifuged at 17,000´g for 30 min to remove the proteins. The supernatants were dried under vacuum, and the residues were dissolved in
methanol. Aliquots were separated by HPLC with a Chemcobond
ODS-W column (Chemco, Osaka, Japan), by using the following program: 20 mM KH2PO4 for 15 min, followed by linear gradient of 0%
acetonitrile and 20 mM KH2PO4 to 80% acetonitrile and 4 mM
KH2PO4 over 30 min. The fractions were collected and dried under
vacuum, and the residues were resuspended in ethanol. After centrifugation to remove any possible salt precipitates, the solutions were subjected to fast atom bombardment mass spectrometry (JMS-SX102 or
Fig. 2 Effect of overexpression of AtIPT4 in Arabidopsis calli. Calli
were co-cultivated with Agrobacterium harboring the control vector
(top) or 35S::AtIPT4 (bottom) for 2 d and then cultured on plates containing 0.3 mg ml–1of the auxin indolebutyric acid (IBA) (left) or
0.3 mg ml–1 IBA and 1 mg ml–1 trans-zeatin (right) for 20 d.
JEOL MStation, JEOL DATUM LTD., Tokyo, Japan). Data obtained
with JEOL MStation are shown.
Results
Search for putative isopentenyltransferase genes
As the first step to identify genes coding for enzymes that
catalyze isopentenylation of adenine nucleotides, the amino
acid sequences of the bacterial DMAPP:AMP isopentenyltransferases, IPT (accession number, AJ237588.1) and TZS
(S03122) of A. tumefaciens; PTZ (CAA34418) of Pseudomonas syringae; and IPT(CAA86510) of Pantoea agglomerans; as well as the DMAPP:tRNA isopentenyltransferases
MiaA (B373178) of E. coli and MOD5 (g1419759) of S. cerevisiae were analyzed. For the sequence analysis, Val, Leu and
Ilu were regarded to be exchangeable each other. Sequence
analysis revealed a consensus pattern of GxTxxGK[ST]xxxxx
[VLI]xxxxxxx[VLI][VLI]xxDxxQx{57,60}[VLI][VLI]xGG
[ST], where x denotes any amino acid residue, [ ] any one of
the amino acid residues in [ ], and x{m, n} m to n amino acid
residues in number.
A pattern-matching search (http://www.arabidopsis.org/
cgi-bin/patmatch/nph-patmatch.pl#syntax) identified a number
of genes that matched the pattern. During this study, the
number of homologous genes in the database increased to nine
with the progression of the Arabidopsis genome project. During the course of this study, I knew that another research group
New cytokinin biosynthetic route
Table 1
activity
Apparent
AMP:DMAPP
isopentenyltransferase
% of AMP that accepted
the isopentenyl moiety a
Test sample
Crude extract b
Purified AtIPT4 c + 2´ binding buffer d
Unbound fraction e + dilution buffer f
Purified AtIPT4 c + unbound fraction e
64.3
0.1
5.2
27.6
a
Percentage of AMP that were converted to iPA through isopentenyltransfer reaction followed by phosphatase treatment (this will be 100%
if all AMP in the reaction mixture is isopentenylated).
b
30 ml of crude extract of E. coli expressing AtIPT4, c 15 ml of AtIPT4
(0.4 mg) with d 15 ml of 2´ binding buffer, e 15 ml of unbound fraction
with f dilution buffer, or c 15 ml of purified AtIPT4 with e 15 ml of
unbound fraction was mixed with 30 ml of a 2´ assay mixture containing 2 mM [2-3H]AMP, and incubated for 1 h at 25°C. After CIAP treatment and ethyl acetate-extraction, radioactivity was measured.
was working on the same set of genes, so we reached an agreement to use the same nomenclature, AtIPT1-9 (Takei et al.
2001, AtIPT9 was excluded in their report), for these genes
(Fig. 1). Of the nine genes, AtIPT6 seems a pseudogene of the
WS strain, because there is a nucleotide deletion that would
cause a frameshift, and no expression of AtIPT6 was detected
by RT-PCR (data not shown). Messages of AtIPT1-5, 7, 8 were
detected. The cDNA sequence for AtIPT9 has been registered
in the database with the accession number AAK25918 indicating that it is expressed at least in the Columbia strain. Expression of AtIPT9 was not tested in the WS strain in this study.
Most of the AtIPT genes had been annotated as tRNA isopentenyltransferases in the Arabidopsis genome project. However, detailed sequence analysis revealed new features. An
amino acid sequence alignment (Fig. 1A) revealed that Arabidopsis isopentenyltransferases, bacterial cytokinin biosynthetic
isopentenyltransferases and tRNA isopentenyltransferases
exhibited significant sequence similarity in their aminoterminal regions. An ATP/GTP binding motif GxxxxGK[TS]
(Saraste et al. 1990) was present in this region (Fig. 1A). The
Table 2
681
Fig. 3 SDS-polyacrylamide gel electrophoresis of recombinant
MiaA, TZS and AtIPT4. MW marker: molecular weight marker.
phylogenetic tree (Fig. 1B) indicates that AtIPT1,3-8 form a
branch. AtIPT2 and AtIPT9 are distantly related to the
AtIPT1,3-8 group and resemble DMAPP:tRNA isopentenyltransferases. There are subgroups in the AtIPT1,3-8 group, one
consisting of AtIPT1,4,6,8 and another of AtIPT3,5,7. Hypothetical rice homologues were also identified in the Monsanto
rice genome database (http://www.rice-research.org/), and other
plant homologues were identified from the expressed sequence
tag database (Fig. 1B).
Effect of over-expression of AtIPT genes
It is known that cytokinins applied exogenously (Skoog
and Miller 1957) or endogenously through expressing the ipt of
Agrobacterium (Ebinuma et al. 1997, Kunkel et al. 1999)
induce cell division and shoot formation on calli. In order to
examine the function of AtIPT genes, AtIPT2 and AtIPT4 were
first selected and introduced into Arabidopsis calli under the
control of the 35S-promoter. Calli transformed with the control
vector exhibited normal hormone responses: root formation in
the presence of only an auxin, and shoot formation in the pres-
Substrate-specificity of isopentenyl transferases
Enzyme
MiaA f
AtIPT4 f
AtIPT4 g
TZS f
No enzyme
DMAPP:tRNA isopentenyltransferase
activity a
71.4
1.5
not tested
1.0
1.0
DMAPP:ATP isopentenyltransferase
activity b
DMAPP:ADP isopentenyltransferase
activity c
DMAPP:AMP isopentenyltransferase
activity d
0.1
75.7
44.8
0.1
0.1
0.1
61.7
33.1
0.3
0.1
0.0
0.1
0.2
90.0
0.0
DMAPP:adenosine isopentenyltransferase
activity e
0.2
0.2
0.5
0.3
0.3
Percentage of [3H]DMAPP (0.67 mM of initial concentration) that transferred the isopentenyl moiety to tRNA.
Percentage of b ATP, c ADP, d AMP, or e adenosine (1 mM of initial concentration) that accepted the isopentenyl moiety from DMAPP.
f, g
Protein concentrations of enzymes were f10 mg ml–1 and g 1 mg ml–1 and reactions were for 10 min.
a
b, c, d, e
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New cytokinin biosynthetic route
Fig. 4 Enzymatic properties of AtIPT4 as a DMAPP:ATP isopentenyltransferase. Reaction velocities were plotted against ATP levels (A) and
against DMAPP levels (B). Lineweawer-Burk plots are shown as an inset.
ence of a cytokinin and an auxin. By contrast, calli transformed with 35S::IPT4 regenerated shoots even in the absence
of exogenously applied cytokinins (Fig. 2). However, there
were no appreciable differences between calli transformed with
35S::IPT2 and calli transformed with the control vector (data
not shown). These results indicated that AtIPT4, and its closely
related gene products, but not AtIPT2, were strong candidates
for cytokinin biosynthetic enzymes.
In vitro isopentenyltransferase activity
To identify enzyme activity of AtIPTs, crude extracts of E.
coli expressing AtIPT2 (data not shown), AtIPT4 or AtIPT5
(data not shown) were first examined for DMAPP:AMP isopentenyltransferase activity. Crude extracts of AtIPT4 and
AtIPT5 possessed this activity, but that of AtIPT2 did not.
However, AtIPT4, which was selected for detailed analysis,
lost this activity after purification (Table 1). All attempts aimed
at retaining the activity during purification were unsuccessful.
However, there was a possibility that E. coli factors that were
not retained on the affinity-purification resin were required for
AtIPT4 to exert the isopentenyltransferase activity. A mixture
of the purified AtIPT4 and the fraction unbound to the affinity
resin possessed high activity, whereas either fraction alone had
no or weak activity. The results indicated that factors that did
not bind to the affinity resin were also required for the purified
AtIPT4 to exert the activity. Possible E. coli-derived factors are
adenylate kinase and ATP, which are present in all organisms,
the former of which catalyzes a reaction: AMP + ATP ¨
ADP. Because E. coli extract should contain both ATP and adenylate kinase, the radiolabeled AMP included in the assay mixtures could have formed radiolabeled ATP and ADP, which
were candidate substrates.
As shown in Table 2, ATP and ADP acted as substrates of
the affinity-purified AtIPT4, which had been purified to near
homogeneity (Fig. 3), but AMP did not at all. The result explicitly revealed that AtIPT4 was DMAPP:ATP/ADP isopentenyltransferase, and had no activity of DMAPP:AMP isopentenyltransferase. In contrast, TZS used AMP as a sole acceptor of
the isopentenyl moiety, as was already reported by Morris et al.
(1993). AtIPT4 also resembles DMAPP:tRNA isopentenyltransferase in its sequence. MiaA of E. coli used tRNA as the
sole acceptor of the isopentenyl moiety in the present assay,
and is indeed a DMAPP:tRNA isopentenyltransferase. AtIPT4
and TZS never used tRNA as the acceptor.
The Km values of AtIPT4 for ATP and DMAPP were estimated to be 18 mM and 6.5 mM, respectively, and the maximum reaction velocity was 0.22 mmol (mg protein)–1 min–1
(Fig. 4), when ATP was used as the isopentenyl acceptor. The
DMAPP:ATP isopentenyltransferase activity required magne-
Table 3 Effect of Mg2+ on the DMAPP:ATP isopentenyltransferase activity of AtIPT4
Mg2+
+b
–c
a
%of ATP that was isopentenylated a
11.2%
0.7%
Percentages of 1mM ATP that accepted the isopentenyl moiety during
4 min incubation.
b
Reactions contained 5 mM MgCl2.
c
Reactions contained 2.5 mM EDTA in place of MgCl2. The concentration of AtIPT4 in reactions was 1 mg (protein ml–1).
New cytokinin biosynthetic route
683
chromatographed on an octadecyl column, two major peaks
were identified. One of these peaks corresponded to the retention time of ATP, but the other did not correspond to the retention times of ATP, adenosine, or iPA (Fig. 5A). However, after
treatment of the reaction products with CIAP, two major peaks
appeared at different retention times (Fig. 5B, peaks 3 and 4).
The retention times of peaks 3 and 4 were the same as those for
adenosine and iPA, respectively. This is consistent with the
hypothesis that peak 2 (Fig. 5A) was isopentenyl ATP (iPTP)
and peak 1 was unreacted ATP.
The identity of possible iPTP was further confirmed by
mass spectrometry. Because the product without phosphatase
treatment did not give a specific peak on mass spectrometry,
probably due to interference of the triphosphate group to ionization, dephosphorylated product (Fig. 5B, peak 4) was analyzed. This provided an [M + H]+ peak of m/z 336, corresponding to the peak of protonated iPA (Imbault et al. 1993). The
observed m/z value of 204 corresponds to a fragmentation ion
peak of iPA (Imbault et al. 1993).
Discussion
Fig. 5 Analysis of the AtIPT4-catalyzed reaction product. (A)
HPLC-elution profile of the isopentenylation reaction product. The
retention time for ATP is indicated by the arrow. (B) Elution profile of
the alkaline-phosphatase-treated isopentenylation reaction product.
Retention times of adenosine and iPA are indicated by arrows. (C)
Positive ion mass spectrum of HPLC peak 4 in (B). Peaks specific for
samples are marked with asterisks (m/z = 204 and 336). Peaks at m/z =
185, 207, 277, 369 were derived from matrix (glycerol).
sium ion for its reaction (Table 3).
Mass spectrometric analysis of the product
To confirm that AtIPT4 catalyzed the transfer of the isopentenyl moiety to ATP, the reaction products were analyzed
by HPLC and mass spectrometry. When the products were
It has been thought that an important step of cytokinin
biosynthesis is the transfer of the isopentenyl group to AMP
(Chen 1997, McGaw and Burch 1995). Despite extensive studies to identify cytokinin biosynthetic isopentenyltransferases of
plants, none has been explicitly identified and characterized.
Through database searches, Takei et al. (2001) and we have
independently identified nine potential isopentenyltransferase
sequences (AtIPT1-9) in Arabidopsis. Out of the nine candidate sequences, seven gene products, AtIPT1 and AtIPT3-8,
have similar sequences and comprise a branch of the phylogenetic tree. They showed that a crude extract of E. coli expressing these genes had DMAPP:AMP isopentenyltransferase
activity. Overexpression of AtIPT4 in Arabidopsis induced
cytokinin responses in cullus culture in the absence of added
cytokinins. However, recombinant AtIPT2 did not exhibit isopentenyltransferase activity when either of ATP, ADP, AMP or
adenosine was used as the acceptor of the isopentenyl moiety
(data not shown). Moreover, 35S-linked ATIPT2 did not induce
cytokinin responses in calli in the absence of cytokinins. The
results of Takei et al. (2001) and results of the present study
indicate that AtIPT1 and AtIPT3-8 are cytokinin synthases of
Arabidopsis.
Takei et al. (2001) reported that the purified AtIPT1 had
DMAPP:AMP isopentenyltransferase activity. The Km values
of AtIPT1 for AMP and DMAPP were 185 and 50 mM, respectively, which seems to be rather high. Furthermore, the ATP/
AMP ratio in eukaryotic cells under non-stressed conditions is
estimated to be ~100 (Hardie et al. 1998). Therefore, a question arises as to whether DMAPP:AMP isopentenyltransferase
activity of AtIPT1 plays a major role in cytokinin biosynthesis
in Arabidopsis. In this paper, the reaction catalyzed by AtIPT4
was characterized in detail, and it was revealed that AtIPT4
684
New cytokinin biosynthetic route
Fig. 6 A model for the cytokinin biosynthetic pathways in plants. In this model, cytokinin biosynthesis is initiated mainly by the addition of isopentenyl side chain to ATP and ADP.
was DMAPP:ATP/ADP isopentenyltransferase, a novel
enzyme not reported so far. The Km values of AtIPT4 for ATP
(18 mM) and DMAPP (6.5 mM) are comparable to those of
TZS (11.1 mM for AMP and 8.2 mM for DMAPP, Morris et al.
1993). A preliminary experiment showed that AtIPT1 also
transferred efficiently the isopentenyl group to ATP and ADP
(data not shown). These results indicated that the major function of AtIPT4, and probably that of AtIPT1, in plant cells are
isopentenylation of ATP and ADP.
A direct proof of the reaction products, iPTP or isopentenyl ADP (iPDP), has been unsuccessful because of the unavailability of authentic iPTP and iPDP for comparison in HPLC
and possible interference of the phosphate group to ionization
in mass-spectrometry. However, a compound which produced
iPA after phosphatase treatment was produced by the reaction
of AtIPT4 with ATP and DMAPP. The result indicates that the
reaction products are phosphorylated iPA. It is highly likely
iPTP and iPDP were produced, when ATP and ADP, respectively, were used as isopentenyl acceptor.
The presence of iPMP-independent pathway to produce
zeatin-type cytokinins was recently demonstrated in Arabidopsis (Åstot et al. 2000). If one of methyl groups of iPTP and
iPDP are hydroxylated to produce ZPT and ZDP, respectively,
followed by dephosphorylation, the ATP/ADP-derived pathway
described here could be an iPMP-independent pathway
reported by Åstot et al. However, it is still possible that there
may be another iPMP-independent pathway, in which hydroxylation of the side chain occurs prior to be transferred to the N6
of the adenine structure.
Although Takei et al. (2001) showed that an E. coli extract
expressing any one of AtIPT1 and AtIPT3-8 exhibited
DMAPP:AMP isopentenyltransferase activity, substrate specificity of this class of enzymes can only be determined with purified enzymes, as was revealed in this study. To establish the
framework of the cytokinin biosynthetic pathways (Fig. 6) in
plants, it is important to determine the substrate specificity of
all isopentenyltransferases of Arabidopsis in purified form, and
to develop methods to identify and quantify iPTP and iPDP in
plants.
Acknowledgements
I thank Hiroshi Adachi of Osaka University and Yoshiaki Sakurai
of the Technology Research Institute of Osaka Prefecture for mass
spectrometry, Miho Matsumoto for technical assistance, Satoru Kawamura for allowing me to use a HPLC facility, and A. Takisawa and M.
Hasebe for valuable discussions. This work was supported in part by
New cytokinin biosynthetic route
JST and grants from the Ministry of Education, Culture, Sports, Science and Technology (grant numbers 09440267, 12142207, and
13073–2125–14).
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(Received May 16, 2001; Accepted June 8, 2001)