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INTRODUCTION |
Cytokinin is a phytohormone involved in various processes of
growth and development of plants, such as cell division,
photosynthesis, chloroplast differentiation, senescence, and nutrient
metabolism (1). Although phenylurea-type species are known (2), the most abundant cytokinins in plants are adenine-type species, which are
adenines substituted at N6 with an isoprenoid or aromatic
side chain.
Multiple routes have been proposed in cytokinin biosynthesis. Transfer
RNA degradation has been suggested to be a source of cytokinin (3),
because some tRNA molecules contain an isopentenyladenosine (iPA)1 residue at the site
adjacent to the anticodon. The modification is catalyzed by tRNA
isopentenyltransferase (tRNA IPT; EC 2.5.1.8), which has been
identified in various organisms such as Escherichia coli
(4-6), Saccharomyces cerevisiae (7, 8), Lactobacillus acidophilus (9), Homo sapiens (10), and Zea
mays (11). However, from the calculated tRNA turnover rate, it is
estimated that the degradation pathway is not a major source of
cytokinin (12). Another possible route of cytokinin formation is
de novo biosynthesis of iPMP by adenylate
isopentenyltransferase (IPT; EC 2.5.1.27) with DMAPP and AMP as
substrates. In the plant pathogenic crowngall-forming bacterium,
Agrobacterium tumefaciens, the IPT gene on the Ti-plamid
(13) is integrated into the genome of host plant cells after infection.
Overproduction of cytokinins by the transduced IPT causes
abnormal cell proliferation. The gene has been identified in various
bacterial species (13-17), and the translated product has been proved
to biosynthesize iPMP, an active cytokinin, in vitro (18).
On the other hand, there is little concrete evidence of the authentic
biosynthesis of iPMP by IPT in higher plants. Cytokinin has been
suggested to be synthesized in specific sites such as the root tip
(19), immature kernel (20), and shoot apical meristem (21). However,
the activity of plant IPT has been reported in only a few tissues such
as immature maize kernels (20) and cytokinin autonomous tobacco callus
(22, 23). As the enzyme seemed to be highly unstable and low in
content, biochemical approaches to purify and characterize plant IPT
have been hampered.
As the first step toward understanding the cytokinin biosynthetic
pathway at the molecular level, we tried to identify IPT genes in
Arabidopsis thaliana. Our study showed that Arabidopsis contains multiple IPT genes encoded by a small multigene family. To our knowledge, this is the first report on the identification of IPT
in a higher plant at the molecular level.
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EXPERIMENTAL PROCEDURES |
Plant Materials--
A. thaliana ecotype Columbia was
grown on vermiculite (24) at 22 °C under fluorescent light, at an
intensity of about 100 µE m
2
s
1, in a growth chamber with a photoperiod of
16 h (day)/8 h (night).
RT-PCR--
Total RNA was prepared by the guanidine thiocyanate
procedure (25). Complementary DNAs were amplified with
SuperScript One-step RT-PCR system (Life Technologies, Inc.)
essentially as described by the supplier. Sequences of the primers
for PCR were: for the AtIPT1, 5'-TCATGACAGAACTCAACTTCCACC-3' and
5'-ATAAAGCTTCTAATTTTGCACCAAATGCCGC-3'; for the AtIPT2,
5'-CGCGGTACCGTCATGATGATGTTAAACCCTAGC-3' and
5'-ATAGTCGACTGATATATAAATCAATTTACTTCTGC-3'; for the AtIPT3,
5'-CGCGGATCCATCATGATCATGAAGATATCTATGGC-3' and 5'-ATAGTCGACGTGGTTACAACTGATCACGCC-3'; for the AtIPT4,
5'-TCATGAAGTGTAATGACAAAATGG-3' and
5'-ATAGTCGACGTTTTGCGGTGATATTAGTCC-3'; for the AtIPT5,
5'-GGGATCATGAAGCCATGCATGACGGC-3' and 5'-GGTTCCTGCAGTACCTCACCGGG-3'; for
the IPT6, 5'-CAACAACTCATGACCTTGTTATCACC-3' and
5'-GGCCAAGCTTGGAAAAACAGACTAAACTTCC-3'; for the AtIPT7,
5'-GGCGGATCCTCATGAAGTTCTCAATCTCATC-3' and
5'-GGCCTGCAGCTTTTCATATCATATTGTGGG-3', and for the AtIPT8, 5'-CAAAATCTTACGTCCACATTCGTCTC-3' and
5'-CCGGCTGCAGCTCACACTTTGTCTTTCACC-3'. The primers were designed to
generate appropriate restriction sites for constructing the expression
plasmids as described below. Reverse transcription was carried out at
50 °C for 30 min, and successive PCR was carried out for 40 cycles
at 94 °C for 0.5 min, 55 °C for 0.5 min, and 70 °C for 1.5 min
in a thermal cycler (RoboCycler, Stratagene, La Jolla, CA). The
products of PCR were subcloned into the plasmid pT7blue T-vector
(Novagen, Madison, WI).
DNA Sequencing and Sequence Analysis--
Sequencing of
cDNAs was performed by the dideoxy chain-termination method (26)
using an ABI-PRISM BigDye terminator cycle sequencing kit
(Applied Biosystems, Foster City, CA) with an automated DNA
sequencer (310 Genetic Analyzer, Applied Biosystems). The GENETYX software system (Software Development Co., Tokyo, Japan) was
used for computer analysis of nucleotide sequences and of deduced amino
acid sequences.
Construction of Expression Plasmids--
Complementary DNAs
containing the reading frame of AtIPT1 to AtIPT8 were excised from the
pT7blue derivatives by digestion with appropriate restriction enzymes.
Each DNA fragment was inserted into pTrc99A vector (Amersham Pharmacia
Biotech) at the NcoI site. The JM109 strain of E. coli was used as the host for protein expression. For the
expression of Agrobacterium IPT (tmr) in E. coli, the reading frame was amplified by PCR with primers:
5'-CGCAAAAAACCCATGGATCTGCGTC-3' and 5'-CGAACATCGGATCCAAATGAAGACAGG-3',
and pTi-SAKURA from A. tumefaciens MAFF301001 (17) as a
template. The amplified DNA was digested with NcoI and
BamHI, and the resulting fragment was ligated into a
pTrc99A vector.
Expression of IPTs in E. coli--
Transformants were grown in
M9 minimal medium, which is supplemented with 20 µg/ml ampicillin, 1 M sorbitol, 1% (w/v) casamino acid, 2% (w/v) sucrose, 2.5 mM betaine, 5 µg/ml thiamine, 1 mM MgSO4, and 0.1 mM CaCl2. The
cultures were incubated at 25 °C with shaking until the
A600 was 0.5. Expression of the
IPTs was induced by incubation with 1 mM IPTG at 25 °C
for 4 h. The cells were harvested by centrifugation, and the
supernatants were used for determination of cytokinin excreted into the
medium by ELISA.
E. coli Plating Assay--
An E. coli strain having
the
rcsC and cps::lacZ genetic
background that had been transformed with pIN-III-AHK4 (27) was cultured in Luria broth. Details for the pIN-III plasmid vector were
described by Masui et al. (28). The culture of the
transformants was mixed with that of E. coli expressing each
AtIPT protein. The mixed cells were spotted on Luria agar plates
supplemented with X-Gal and incubated at 25 °C.
Purification of Recombinant AtIPT1 from E. coli Cells--
The
bacterial cells obtained from 1 litter of culture was suspended with
buffer A (1 M betaine, 20 mM HEPES, 100 mM KCl, 10 mM MgCl2, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride,
pH 7.5). The cells were disrupted by ultrasonic irradiation on ice. The homogenate was centrifuged at 15,000 × g for 20 min at
4 °C. Nucleic acids were removed from the supernatant by
precipitation with 0.0625% (w/v) protamine sulfate. After
centrifugation, the supernatant was diluted with an equal volume of
buffer B (1 M betaine, 20 mM HEPES, 5 mM MgCl2, 1 mM DTT pH 7.5) and
loaded onto a column of Mono S (FPLC system; Amersham Pharmacia
Biotech) that had been equilibrated with buffer B. The column was
eluted with a linear gradient of KCl from 0 to 500 mM.
Pooled fractions containing IPT activity were loaded on a Superdex
200-pg column (HiLoad 16/60, FPLC system) in buffer C (1 M
betaine, 20 mM triethanolamine, 100 mM KCl, 10 mM MgCl2, 1 mM DTT, pH 8.0). The
final preparation of the AtIPT1 fraction was divided into 0.1-ml
aliquots and stored at
80 °C.
Enzyme Assays for IPT--
Two methods were applied to measure
the IPT activities. (i) Radioisotope rapid assay: the enzyme assay was
carried out as essentially described by Blackwell and Horgan (18). (ii)
Nonradioisotope assay: enzyme was incubated in a reaction mixture (1 M betaine, 20 mM triethanolamine, 50 mM KCl, 10 mM MgCl2, 1 mM DTT, 1 mg/ml bovine serum albumin, pH 8.0) with 1 mM AMP and 340 µM DMAPP at 25 °C for 20 min. The reaction was stopped by the addition of a quarter volume of
10% acetate and centrifuged at 18,000 × g for 20 min.
The resulting supernatant was subjected to HPLC with an ODS column
(Merck, Supersphere RP-select B; 4 mm inside diameter × 250 mm).
Other conditions were as described previously (29). One unit of IPT
activity was defined as the amount of enzyme that produced 1 µmol of
iPMP/min under the condition of the reaction.
Identification of Cytokinin Species by Mass
Spectrometry--
Liquid chromatography-mass spectrometry analysis of
cytokinins was performed on a Platform II LC-MS (Jasco, Tokyo, Japan) with a C18 column (Wakosil-II 5C18 RS, 1 mm inside
diameter × 250 mm) using a positive ion electrospray ionization.
The cone voltage was 42 V, source temperature was 70 °C, and
capillary voltage was 3.0 V. Data were analyzed using Masslinx version
2.1 software.
Others--
Protein was quantitated by Bradford's method (30)
with bovine serum albumin as the protein standard. The conventional
techniques for manipulation of DNA were those described by Sambrook
et al. (31). SDS-PAGE was performed by the method of Laemmli
(32).
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RESULTS |
Isolation of a Set of cDNAs Encoding IPT-like Proteins--
To
pick up plant gene(s) encoding IPT, we screened the genome sequence of
A. thaliana in silico against the amino acid sequence of an
Agrobacterium IPT, tmr (17), as queries. Consequently, eight
candidates, designated as AtIPT1 to AtIPT8 having
significant homology to tmr, were found. At the amino acid
level, tmr has 37.3% similarity to AtIPT1, 32.8% to AtIPT2, 40.8% to
AtIPT3, 44.7% to AtIPT4, 40.9% to AtIPT5, 41.2% to AtIPT6, 42.2% to
AtIPT7, and 43.6% to AtIPT8. Table I
summarizes the structural features of the sequences. The AtIPT genes
are distributed all over five chromosomes of Arabidopsis. AtIPT2 was
equivalent to a sequence registered as Arabidopsis tRNA IPT
(GenBankTM accession numbers AAF00582 and AF109376 for the
protein and the mRNA, respectively). Six of the eight (AtIPT1,
AtIPT3, AtIPT4, AtIPT6, AtIPT7, and AtIPT8) have been deposited and
annotated as "putative" tRNA IPT based on the sequence
similarities. One was not annotated as an open reading frame, but we
found a possible reading frame on chromosome V, which has homology to
other AtIPTs, and designated it AtIPT5.
To obtain the cDNAs, total RNA was prepared, and RT-PCR was
performed with specific primers as described under "Experimental Procedures." Each PCR amplified a specific cDNA fragment with an
expected length (data not shown), and the nucleotide sequences of the
cDNAs were determined. Fig.
1A shows a sequence comparison of a set of deduced amino acid sequences of AtIPT proteins. The reading
frames of AtIPTs deduced from the cDNA sequences consisted of
318-466 amino acids, which have 34.7-60.6% amino acid identities to
AtIPT1. AtIPT2 encoded the longest reading frame. Multiple alignment of the AtIPT showed that AtIPT2 contains two inserted regions
of about 80 and 20 amino acids. The carboxyl-terminal region of AtIPT2
also had an extra 40 amino acids. Fig. 1B shows a
phylogenetic tree based on a comparison with other representative IPT
and tRNA IPT sequences. This calculation implies that the divergence of
the AtIPT2 gene occurred before that of other
AtIPTs and bacterial IPT genes. These results suggest that
the primary structure of putative AtIPT proteins, AtIPT1 and AtIPT3 to
AtIPT8, are more closely related to bacterial IPT than tRNA IPT.

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Fig. 1.
Multiple alignment of amino acid
sequence of the predicted translation products of
AtIPTs (A) and phylogenetic tree of
representative tRNA IPTs and IPTs (B).
A, gaps denoted by dashes were inserted to obtain
maximum homology. The identical amino acid residues among all AtIPTs
are indicated by white letters on a black
background and those with AtIPT1 are hatched. Marked
stretches (Regions a and b) are discussed in the
text. B, the tree was generated using the CLUSTALW program
at the DNA Data Bank of Japan. Relative branch lengths are
approximately proportional to phylogenetic distance. Eukaryotic tRNA
IPT (magenta) from S. cerevisiae
(GenBankTM accession number P07884);
Schizosaccharomyces pombe (Gen- BankTM
accession number CAB52278); H. sapiens
(GenBankTM accession number AF074918); C. elegans (Gen- BankTM accession number
T27538), prokaryotic tRNA IPT (cyan) from Aquifex
aeolicus (GenBankTM accession number G70391);
Borrelia burgdorferi (GenBankTM accession
number AAC67163); Richettsia prowazekii
(Gen- BankTM accession number CAA14962);
Mycobacterium leprae (GenBankTM accession
number S72942); Streptomyces coelicolor
(GenBankTM accession number T35111); A. tumefaciens (GenBankTM accession number
P38436); Deinococcus radiodurans (GenBankTM
accession number AAF11245); E. coli
(GenBankTM accession number AAC77128);
Pseudomonas putida (Gen- BankTM accession
number AAB69443); Thermotoga maritima
(GenBankTM accession number C72366);
Bacillus subtilis (GenBankTM accession
number G69657); Chlamydia trachomatis
(GenBankTM accession number AAC68361);
Synechocystis sp. PCC6803 (GenBankTM accession
number S75554), bacterial IPT (green) from
Agrobacterium rhizogenes pRiA4 (GenBankTM
accession number S06738); A. tumefaciens pTiC58
(GenBankTM accession number AAA27406); A. tumefaciens pTi-SAKURA (17); Agrobacterium vitis pTiS4
(GenBankTM accession number S30106);
Pseudomonas syringae pCK1 (GenBankTM accession
number A24937); Pseudomonas solanacearum
(GenBankTM accession number S06739), and
Rhodococcus fascians pFiD188 (GenBankTM
accession number CAA82744).
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Expression of IPTs in E. coli--
The eight cDNAs,
AtIPT1 to AtIPT8, were expressed in E. coli under the control of the trc promoter, which can
be driven by IPTG. First, to examine the ability of the expressed
protein to synthesize cytokinin in vivo, we measured the
cytokinin content in the culture medium. As the substrates of IPT,
DMAPP, and AMP are provided by authentic metabolism in E. coli, we expected the cytokinins to be synthesized and excreted.
As a control, tmr from A. tumefaciens pTi-SAKURA
(17) was also expressed in E. coli. As shown in Fig.
2A, when tmr
expression was induced, iP was predominantly accumulated in the culture
medium. A small amount of Z was also detected. Expression of
AtIPT1 and AtIPT3 to AtIPT8 also
caused the accumulation of iP and Z in the media. In the culture of
E. coli transformants of AtIPT1,
AtIPT4, AtIPT7, and AtIPT8, Z content was relatively higher than those of AtIPT3,
AtIPT5, and AtIPT6. iPMP, the possible reaction
product, and other cytokinin species were below the detectable level in
every culture (data not shown). On the other hand, in the culture media
of transformants of AtIPT2, no significant accumulation of
cytokinin species was detected.

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Fig. 2.
Excretion of cytokinin from recombinant
E. coli harboring IPT genes and the IPT activity of
the cell extracts. A, each transformant was cultured in
the presence of 1 mM IPTG in modified M9 minimal medium for
4 h. The culture media of the transformants were collected, and
the cytokinin fraction was purified and the molecular species
determined by ELISA. Values shown are the means of three independent
replicates. B, the culture of E. coli strain
( rcsC, cps::lacZ) harboring
pIN-III-AHK4 (27) was mixed with that of E. coli expressing
each AtIPT. The mixed cells were spotted on Luria agar
plates supplemented with X-Gal and incubated at 25 °C for 48 h.
C, the crude extract of each transformant cell was used to
measure the IPT activity by radioisotope rapid assay. The
amount of each sample for assay was equivalent to 1 A600 unit of cells. One
A600 unit is defined as the amount of
cells obtained from 1 ml of cell culture whose
A600 value is 1.
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Using an E. coli system, we attempted to examine the
cytokinin biosynthesis ability of the transformants of
AtIPT. In Arabidopsis, a cytokinin receptor, AHK4 (identical
to CRE1), has been identified recently (27, 33). The Rcs-phosphorelay
system in E. coli (RcsC
YojiN
RcsB) is involved in
extracellular polysaccharide synthesis by activating the cps
operon (27). In the E. coli strain having the
rcsC and cps::lacZ genetic
background, AHK4 can function as a cytokinin-responsive sensory
His-kinase through activating the E. coli
YojN
RcsB
cps::lacZ pathway, thereby giving rise to blue colonies in the presence of external cytokinin and X-Gal
(27). When each of the transformants of AtIPT was mixed with
that of AHK4 and grown in the presence of X-Gal, all those except for that of AtIPT2 turned blue without externally
added cytokinin (Fig. 2B). This was well consistent with the
result shown in Fig. 2A.
To confirm the ability of the gene products to synthesize cytokinin,
IPT activity was measured by the radioisotope rapid assay with the
total extract of the E. coli transformants (Fig.
2C). Although the extent was different, IPT activity was
detected in all extracts of the transformants of AtIPTs
except for AtIPT2. These results suggest that the gene
products of AtIPTs other than AtIPT2 could
synthesize cytokinin species in E. coli cells.
Purification and Characterization of Recombinant AtIPTs--
To
confirm further the catalytic reaction of the recombinant protein, the
AtIPT1 polypeptide was purified as described under "Experimental
Procedures." SDS-PAGE of the final preparation showed it to be
apparently homogeneous (Fig. 3,
lane 6). The catalytic activity of IPT was determined by a
nonradioisotope assay.

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Fig. 3.
Purification of AtIPT1 produced in E. coli. Samples (lanes 1-4, 20 µg; lanes 5 and 6, 5 µg) from various purification stages were
subjected to SDS-PAGE. Lane 1, total extract of noninduced
E. coli; lane 2, total extract of 4-h-induced
E. coli; lane 3, soluble fraction of the induced
E. coli; lane 4, supernatant of protamine sulfate
precipitation; lane 5, Mono S column chromatography
fraction; lane 6, Superdex 200-pg fraction. The gel was
stained with Coomassie Brilliant Blue. The molecular masses of marker
proteins are indicated in kilodaltons (kDa) on the
left.
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Purified AtIPT1 was incubated with AMP and DMAPP, and the products were
loaded on HPLC (Fig. 4). The retention
time of a peak of one of the reaction products (Peak A) was
identical to that of a peak of iPMP (Fig. 4, A and
B). When the reaction products were treated with alkaline
phosphatase, the retention time of Peak A was shifted to that of Peak B
whose retention time coincided with that of iPA (Fig. 4, A
and C). Peaks A and B had an absorbance spectrum identical
to that of iPMP and iPA, respectively (data not shown). To identify
Peak B, mass spectrometry analysis was performed. Consequently, the
mass of the product coincided with the iPA standard (Figs. 4,
D and E). These results clearly demonstrated that
recombinant AtIPT1 catalyzes the IPT reaction.

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Fig. 4.
Identification of reaction products of assay
with purified recombinant AtIPT1. Patterns of elution of iPMP and
iPA (A), reaction products of the nonradioisotope assay with
AtIPT1 (B), dephosphorylated products after treatment with
alkaline phosphatase (C) are shown. iPA standard
(D) and Peak B fraction (E) were subjected to
mass spectrometry.
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Kinetic Parameters of AtIPT1--
The recombinant enzyme of AtIPT
enabled us to analyze the kinetic parameters (Table
II). The specific activity of AtIPT1 was 57 milliunits/mg of protein, and the Km values for
AMP and DMAPP were 185 and 50 µM, respectively. The
Km value for AMP of AtIPT1 was much higher than that
of ipt1 (85.7 nM) in A. tumefaciens (18).
Adenine, adenosine, and isopentenylpyrophosphate were not utilized as
the substrates (data not shown). On the other hand, ATP, GTP, ADP, and
GDP strongly inhibited the IPT activity. The optimum pH was around 8.0, and there was no activity at pH 6 (data not shown). Due to the
unstableness of the other recombinant AtIPTs, the yields of the
purified preparation were quite low (data not shown). The enzymatic
property of them could not be determined.
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DISCUSSION |
In this study, we identified genes encoding IPT, a cytokinin
biosynthesis enzyme, in A. thaliana. The identity of the
cDNA was established by determination of the catalytic activity of the translated products in vivo and in vitro
(Fig. 2) and by chemical determination of the reaction product by mass
spectrometry (Fig. 4). While all AtIPT sequences had been annotated as
putative tRNA IPTs in the A. thaliana Annotation Data
base, our results demonstrated that all AtIPTs except for AtIPT2
have IPT activity.
Although AtIPTs did not show close similarity to tRNA IPTs and
bacterial IPTs at the amino acid level (detailed alignment is not
shown), some common structural features were found. The putative motif
for DMAPP binding (34), which is similar to the ATP/GTP-binding motif
at the amino-terminal region ((A, G)-X4-G-K-(S, T); Fig. 1A, Region a), was conserved in both
types of isopentenyltransferase. In tRNA IPT in E. coli,
some nucleotides such as GTP, ATP, and CTP inhibit the activity in a
competitive manner with respect to DMAPP (34). In this study,
nucleotides such as ATP and ADP strongly inhibited the IPT activity
(Table II), suggesting that these nucleotides inhibit the activity by
competitive access to the DMAPP-binding site and that the energy status
in the cell is involved in the regulation of the IPT activity. Another
structural feature is that the carboxyl-terminal extension of AtIPT2
contained putative a zinc finger-like motif
(C-X2-C-X12,18-H-X5-H;
Fig. 1A, Region b), which is conserved in
eukaryotic tRNA IPT such as H. sapiens (10),
Caenorhabditis elegans (GenBankTM
accession number T27538), and others. The motif is also found in the
murine RNA-binding protein ZFR (35) and thought to play an important
role in the expression and/or the retention of the activity of
eukaryotic tRNA IPTs (10, 36, 37). The absence of IPT activity of
AtIPT2 and the structural similarities between AtIPT2 and tRNA IPTs are
well consistent with that AtIPT2 is registered as tRNA IPT.
The cytokinin species excreted from the E. coli
transformants of AtIPTs did not coincide with that
determined by chemical analysis of the in vitro reaction
product (Figs. 2 and 4). This discrepancy of detected products between
the culture medium and in vitro reaction is
attributed to the metabolization of cytokinins in E. coli
cells. Namely, iPMP synthesized by AtIPTs is metabolized to iP
and Z and excreted to the culture medium. Nonpolar compounds such as iP
and Z are expected to penetrate easily across the cell membrane. In
fact, the E. coli transformants expressing AtIPT1 and AtIPT3 to AtIPT8 had a growth rate
significantly slower than the AtIPT2 (data not shown)
probably due to metabolic depletion of DMAPP in the E. coli.
The tendency was more remarkable in those expressing AtIPT1,
ATIPT4, AtIPT7, and AtIPT8 (data not
shown), which excreted Z into the medium. Further metabolization of
synthesized iPMP to iP and Z by the authentic dephosphorylation,
hydroxylation, and deribosylation systems occur in the E. coli cells.
The existence of isoforms of AtIPT leads us to speculate the
differentiation of the physiological function of each isoform in terms
of the expression site and the regulatory manner. For instance,
cytokinin has been suggested to be synthesized in restricted sites in
which cell proliferation is active (19-21). In terms of gene
regulation, iPMP has been shown to rapidly accumulate in roots in
response to nitrate replenishment to the nitrogen-depleted maize (29).
These data imply that the cytokinin biosynthesis genes are
expressed differently in spatially and temporally specific areas and in
response to various environmental stimuli. Further comparative
analysis of the expression pattern of each gene should help elucidate
the physiological function.
Recently, an alternative pathway for cytokinin biosynthesis has been
proposed by Åstot et al. (38). They provided evidence that
IPT could use an unknown compound of terpenoid origin as a side chain
donor instead of DMAPP, and the initial product of the pathway is
trans-zeatin 5'-monophosphate. When the possible donor compounds become available, we need to determine whether AtIPT
can catalyze the alternative reaction to elucidate the biochemical entity of the alternative cytokinin biosynthesis pathway.