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INTRODUCTION |
A signature motif thought to be involved in the binding of the UDP
moiety of sugar nucleotides has been identified in a wide range of
enzymes that catalyze the transfer of glucose or glucuronic acid to
small molecule acceptors (1). Using the motif to screen sequences in
the genome and expressed sequence tag data bases of
Arabidopsis, a large multigene family has been defined (2). In plants, the conjugation of glucose to small hydrophobic molecules can lead to the formation of glucose esters or glucosides. The former
are high energy compounds and have long been regarded as biosynthetic
intermediates (3), whereas glucosides are generally considered to
represent the storage forms of the aglycones (4). Both transfer
reactions are suggested to occur in the cytoplasm of cells (5), with
the attachment of glucose providing access to membrane transport
systems and passage into either the vacuole (6) or the extracellular
space (7).
It has been known for many years that plant hormones can be
glycosylated, although the role of the conjugates is not well defined
(8, 9). Despite the wide occurrence of glycosylated conjugates
(10-12) and the many studies of plant glycosyltransferases involved in
hormone conjugation (13-15), only three genes encoding hormone
glycosyltransferases have ever been identified, the iaglu gene of maize (16) and two zeatin glycosyltransferases (17, 18). The
maize iaglu gene encodes a UDP-glucosyltransferase (UGT)1 that forms the
1-O-indole acetyl glucose ester (IAGlc), the glucosylated conjugate of indole-3-acetic acid (IAA).
IAA plays a central role in most, if not all, aspects of a plant's
existence, providing a means of transducing external environmental changes into internal adaptive responses. The hormone is known to exist
as the free acid and in conjugated form linked to a wide variety of
compounds such as amino acids, peptides, and sugars (10). Many studies
have used maize as a model to analyze IAA conjugates. In this species,
the glucose conjugate is found in small amounts in vegetative tissue
and has been shown to be an intermediate in the biosynthesis of
IAA-myoinositol (19), a putative intercellular transport form of IAA
from endosperm to shoot (20). The myoinositol conjugate has been found
to be further glycosylated to form IAA-myoinositol galactoside and
IAA-myoinositol arabinoside (21). All of these conjugates of IAA have
been identified in maize seeds and are thought to represent storage
forms, which upon hydrolysis could release free IAA to the seedling
(22). In Arabidopsis, the principal conjugates found in
vegetative tissue are the amide-linked conjugates (90%). The
ester-linked conjugates are also found, but in much smaller amounts
(10%). IAGlc makes up about 34% of this ester pool in 12-day-old
plants (23). The IAA conjugates are suggested to be storage forms of
IAA (24, 25) or catabolism products (26).
Clearly, the field of IAA conjugation is complex and, while many of the
enzymes involved in these interconversions have been partially
purified, the identification of their corresponding genes would open up
the possibility of taking a genetic route to understanding the events
that occur and their consequences in the plant. Although the
iaglu gene of maize was identified 6 years ago, its product
was neither purified nor characterized biochemically, and no further
studies on the gene have been undertaken. Despite the availability of
the iaglu for cross-hybridization and homology searching
within the Arabidopsis genome, no corresponding gene product
with demonstrable IAA conjugating activity has been published to date.
The availability of a detailed phylogenetic analysis of the
Arabidopsis UGT multigene family has now made it feasible to
screen likely family members for IAA-UGT activity. This study
identifies an Arabidopsis gene encoding an IAA-UGT and
provides detailed biochemical analysis of the gene product.
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EXPERIMENTAL PROCEDURES |
Plant Materials--
Wild-type Arabidopsis, ecotype
Columbia, were grown in Levington's seed and modular compost in a
controlled environment of 16/8 h light-dark cycle (22 °C, 220 microeinsteins
2 s
1
light, 18 °C, dark). After 4 weeks, tissue from leaf, stem (all vertical nonleaf or nonflower tissue), root, and inflorescence (flowers
at all stages of development including siliques) were harvested for RNA
extraction. 6-Week-old tissue was used to analyze the steady-state
levels of mRNA in siliques. Young siliques were the first nine from
the tip, and all of the subsequent siliques were classified as old.
Recombinant UGT Purification--
Escherichia
coli strain XL-1 Blue carrying the recombinant GST-UGT
protein expression plasmid (27) was grown at 20 °C in 75 ml
of 2× YT medium containing 50 µg/ml ampicillin until the A600 reached 1.0, after which the culture was
incubated with 1 mM
isopropyl-1-thio-
-D-galactopyranoside for 24 h at
20 °C. The cells were harvested by centrifugation at 5,000 × g for 5 min and were resuspended in 2 ml of Spheroblast
buffer (0.5 mM EDTA, 750 mM sucrose, 200 mM Tris-HCl, pH 8.0) (28). Lysozyme (1 mg) and 14 ml of
half-strength Spheroblast buffer were added immediately. After
incubation at 4 °C for 30 min, the cells were harvested again by
centrifugation and osmotically shocked in 5 ml of phosphate-buffered saline containing 0.2 mM phenylmethylsulfonyl fluoride.
Cell debris was removed by centrifugation at 10,000 × g for 15 min. The protein in the supernatant fraction was
collected by adding 100 µl of 50% glutathione-coupled Sepharose gel
(Amersham Pharmacia Biotech) and recovered in elution buffer (20 mM reduced form glutathione, 100 mM Tris-HCl,
pH 8.0, 120 mM NaCl), according to the manufacturer's instructions. The protein assays were carried out with Bio-Rad Protein
Assay Dye using bovine serum albumin as reference. The purified
recombinant proteins were also analyzed by SDS-PAGE following the
methods described by Sambrook et al. (29).
Glucosyltransferase Activity Assay--
The general
glucosyltransferase activity assay mix (200 µl) contained 2 µg of
purified recombinant proteins, 14 mM 2-mercaptoethanol, 2.5 mM UDPG, 1 mM IAA, 50 mM Tris-HCl,
pH 7.0. The reaction was carried out at 30 °C for 1 h and
stopped by the addition of 20 µl of trichloroacetic acid (240 mg/ml).
The reaction mix was analyzed using the HPLC method.
HPLC Analysis--
Reverse phase HPLC was performed with a
Waters HPLC system (Waters Separator 2690 and Waters Tunable Absorbance
Detector 486; Waters Ltd., Herts, UK) and a Columbus 5-µm
C18 column (250 × 4.60 mm; Phenomenex). A linear
gradient with increasing methanol (solvent B) against distilled
H2O (solvent A) at a flow rate of 1 ml/min over 30 min was
used to separate the glucose conjugates from their aglycone. Both
solvents contained 0.01% H3PO4 (pH 3.0). The
following elution conditions were used: IAA, 10-48% B,
detection 230 nm; IBA, IPA, NAA, 5-OH-IAA, and 2-ox-IAA,
10-70% B,
detection 230 nm; 2,4-dichlorophenoxyacetic
acid and pichloram, 10-100% B,
detection 287 nm. The
cinnamic acids were analyzed under the conditions described previously
(27).
Identification of the 1-O-Glucose Ester Using Gas
Chromatography-Mass Spectrometry--
The reaction was carried out in
200 µl of 100 mM HEPES/NaOH, pH 7.0, containing 14 mM 2-mercaptoethanol, 5 mM MgCl2,
10 mM KCl, 5 mM UDPG, 1 mM IAA.
After 1 h of incubation (or 6 h for isomer analysis) with 5 µg of the enzyme at 37 °C, the reaction was stopped with 1 ml of
acetone. Subsequently, samples were centrifuged, and the supernatant
was dried in vacuo. The product of the reaction was then
separated from the substrates using two Isolute SPE columns, SAX (anion
exchanger) and Env+ (hydrophobic), arranged in tandem. Eluate from the
Env+ column was dried, dissolved in 500 µl of acetonitrile, and
derivatized with
N,O-bis[trimethylsilyl]trifluoroacetamide/trimethylchlorosilane (Pierce), and 1 µl was injected into the gas chromatography-mass spectrometry system. Half of the sample for isomer analysis was treated
with hydroxylamine prior to the silylation with
N,O-bis[trimethylsilyl]trifluoroacetamide/trimethylchlorosilane. The column used for analysis was a 15-m × 0.25-mm Chromopack
CP-SIL 24CB, and injector temperature was set to 280 °C. After the
injection (splitless), the column temperature was held at 80 °C for
2 min and then increased at the rate of 20 °C/min to 200 °C, and
4 °C/min to 280 °C. Column effluent was introduced into the ion
source of a JEOL JMS-700 mass spectrometer. Ion source and a gas
chromatography interface were held at 270 °C. Ions were
generated with 70 eV at an ionization current of 300 µA. A mass
spectrometer operated in full scan mode with an acceleration voltage of
10 kV, scan range between m/z 40 and 800, and
ion multiplier set to
1.2 kV.
Coupled Enzyme Assay--
The IAA-UGT activity was determined as
the release of UDP, which can be measured using a coupled assay
containing UGT, pyruvate kinase, and lactate dehydrogenase (30). The
reaction mechanisms are shown as the following.
The reaction mix, in a total volume of 1.0 ml, contained 50 mM HEPES-NaOH, pH 7.6, 2.5 mM
MgSO4, 10 mM KCl, 0.15 mM NADH, 2.0 mM phosphoenol pyruvate, 10 µl of UGT solution (diluted
into 50 mM HEPES-NaOH, pH 7.6), 3.0 units of pyruvate
kinase, and 4.0 units of lactate dehydrogenase. The coupled enzyme
assay was analyzed over the range 0-5 mM UDPG and 0-1
mM IAA together with a control at the same concentration of
UDPG but with no IAA nor UGT. The change of NAD+ was
detected at 340 nm, and the reaction rate was converted to the unit
millikatals kg
1 using the extinction
coefficient 6.22 × 103
M
1 cm
1
for NADH.
Phospholipid Preparation and Binding Assay--
Phospholipid
vesicles were prepared using the sucrose solution method (31). A
100-µl bed volume of phospholipids (Sigma) was homogenized in
distilled H2O, followed by centrifugation for 5 min at
12,000 × g. the pellet was resuspended in 240 mM sucrose and was incubated at room temperature for 2 h. The liposomes were harvested by centrifugation and then resuspended
in 100 µl in binding buffer (50 mM HEPES, pH 7.6, with or
without 1 mM CaCl2, 1 mM
MgCl2). An aliquot of 10 µl was used for inhibition
assays, and 80 µl was used for binding assays.
UGT84B1 or a recombinant tomato annexin Rp35 (32) was incubated for 15 min at room temperature with liposomes in the presence of binding
buffer. Nonbound protein in the supernatant was separated from the
liposomes by centrifugation at 12,000 × g for 10 min. The supernatant (100 µl) was added to 20 µl of SDS loading buffer. The pellet was washed in 5 volumes of binding buffer, and 100 µl of
50 mM HEPES containing 5 mM EDTA was added to
elute protein bound to the liposomes. After centrifugation, the
supernatant containing EDTA-eluted protein was added to 20 µl of SDS
loading buffer. All of the samples were subsequently boiled for 5 min, and 25 µl of each sample was analyzed by 10% SDS-PAGE.
Reverse Transcription-PCR--
Total RNA was extracted from
plant tissue by the hot phenol method (33) and quantified by
spectrophotometry. The integrity was checked by formaldehyde-agarose
gel electrophoresis (29). 15 µg of each sample was then treated with
RNase-free RQ1 DNase (Promega) at 37 °C for 30 min and terminated
according to the manufacturer's instructions. RNA was then extracted
with phenol and chloroform, and the aqueous phase was precipitated with
ethanol. The integrity of the RNA was again checked by formaldehyde
agarose gel electrophoresis. Reverse transcription was carried out
using a superscript preamplification system (Life Technologies, Inc.) for first strand cDNA synthesis according to the manufacturer's instructions using an oligo(dT) primer. UGT84B1-specific PCR was then
performed using primers 5'-CGGCATATGATGGGCAGTAGTGAGGGTC-3' (5' for
84B1) and 5'-CGGGTCGACGGCGATTGTGATATCACTAATG-3' (3' for 84B1). The PCR
(100 µl) was set up by mixing 2 µl of first strand cDNA, 1×
PCR buffer (Promega), 1.5 mM MgCl2, 0.5 µM each primer, 0.25 mM each deoxynucleoside
triphosphate, and 5 units of Taq DNA polymerase (Promega).
The PCR was carried out in an MJ Research PTC-200 thermal cycler with 5 min at 94 °C, followed by 30 cycles of 1 min of denaturation at
94 °C, 2 min of annealing at 62 °C, and 4 min of extension at
74 °C. The reaction was completed by one cycle of 5 min at 74 °C.
The PCR products were then analyzed by 1% (w/v) agarose gel. After
separation, the PCR products were transferred onto nylon membrane and
probed with a radiolabeled UGT84B1 DNA fragment (29).
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RESULTS |
Identification of UGT Activity toward IAA--
Earlier work
suggested that only one subgroup of putative glucosyltransferase
sequences in the Arabidopsis genome encodes enzymes forming
glucosyl esters (2, 27). This subgroup was analyzed for activity in
synthesizing the glucose ester of IAA. The sequences were cloned
and used to produce soluble recombinant fusion proteins with GST in
Escherichia coli (Fig.
1A). Following purification,
some of these fusion proteins proved to be particularly unstable,
releasing GST (26 kDa) as a separate polypeptide. This is a common
observation with this fusion system (34). Each of these 10 recombinant
proteins was screened for putative UGT activity toward IAA including
the protein corresponding to the previously annotated IAA-UGT in
Arabidopsis data bases (35) (Fig. 1B). Only four
out of all of the sequences consistently produced a more hydrophilic
compound eluting more rapidly in the methanol gradient. The data shown
correspond to an assay time of 1 h at 30 °C to visualize the
trace activities seen for UGT75B1, UGT75B2, and UGT84B2. The major
activity was always observed with UGT84B1, which reached equilibrium
rapidly and did not go beyond 32% conversion under any of the
conditions assayed. A pH profile of enzyme activity indicated a broad
optimum from pH 6 to 7.5 (data not shown). Under conditions described
above, in which the assay was stopped by the addition of
trichloroacetic acid, only a single peak of product was observed, and
this comigrated on HPLC with a known standard of IAGlc (Fig.
1B). However, spontaneous nonenzymatic rearrangement into
the 2-O, 4-O, and 6-O glucose esters
of IAA (30) could be observed when the reaction was not stopped by
trichloroacetic acid addition (Fig. 1C). These multiple
peaks were found to be hydrolyzable by 1 N NaOH (Fig.
1C), and all, as shown in Fig. 1D, contained
radioactive glucose from UDP-[U-14C]glucose.

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Fig. 1.
Identification of an Arabidopsis
IAA UDP-glucosyltransferase. A, proteins purified
from E. coli expressing 10 putative UGTs as GST fusions were
analyzed using 10% (w/v) SDS-PAGE. The proteins were visualized by
Coomassie staining. The phylogenetic relationship of the corresponding
UGT sequences determined by Phylip is also shown above the
gel (2). B, the recombinant UGTs were incubated with IAA (1 mM IAA, 2.5 mM UDPG, 50 mM
Tris-HCl, pH 7.0, 14 mM 2-mercaptoethanol, 2 µg of UGT
for 1 h at 30 °C), and the enzyme activities were analyzed
using HPLC, together with an authentic IAGlc standard. The HPLC
gradient was from 10 to 48% MeOH in 0.01%
H3PO4 over 30 min. C, a standard
reaction mix with 5 µg of UGT84B1, 5 mM UDPG with no
further treatment ( TCA) is compared with that incubated
with 1 N NaOH for 1 h at room temperature
(+NaOH) and that containing 20 µl of trichloroacetic acid
(+TCA). Multiple peaks (1-4) are observed in the
absence of trichloroacetic acid. D, the UV trace
(upper) and radioactivity trace (lower) for
UGT84B1 following standard incubation in 1 mM UDPG and 5 µl of UDP-D-[U-14C]glucose (270 mCi of
mmol 1 and 25 µCi of
ml 1) for 3 h at 30 °C are compared.
The HPLC gradient was from 0 to 48% MeOH over 40 min.
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The mass spectrum of the main reaction product (Fig.
2) was identical to the previously
published spectrum (36) and the spectrum of synthetic IAGlc (37).
Transitions from the molecular ion (m/z 697) to
m/z 653 (expulsion of CO) and
m/z 450 (hydrogen rearrangement and cleavage of
the ester linkage) are specific for glucose 1-O esters. Ions
of m/z 229 and 247 indicate the presence of the
ester-linked indole-3-acetyl group, while the ion series of
m/z 147, 191, 204, 217, 271, and 361 is
characteristic for the glucosyl part of the molecule. Three isomers
were also identified as 2-O-, 4-O-, and
6-O-(indole-3-acetyl)-D-glucose, and the
position of IAA linkage was verified by analysis of the corresponding
oximes (data not shown).

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Fig. 2.
Gas chromatography-mass spectrometry analysis
of the major product of UGT84B1 following incubation with IAA. The
glucose conjugate of IAA corresponding to peak 1 in Fig. 1B was further purified and analyzed by gas
chromatography-mass spectrometry. The identity of this compound was
confirmed to be 1-O-indole acetyl glucose ester.
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Relative Activity of UGTs with Different Substrates--
Enzyme
activity was first compared using two assays: one based on HPLC and a
second based on a coupled assay in which NADH oxidation was followed.
Both methods gave comparable results (data not shown). However, the use
of the coupled assay can avoid the establishment of equilibrium of the
glucosyltransferase reaction, so it was employed in most of the
following studies. The data shown are those for the intact fusion
protein; however, removal of GST from the recombinant UGT was found to
have no effect on enzyme activity (data not shown). A range of
compounds with similar structure to IAA (Fig.
3) were tested as potential substrates for both UGT84B1 and UGT84B2, UGT75B1, and UGT75B2 that had also been
shown to have trace activities against IAA. As shown in Table I, at a substrate concentration of 1 mM, UGT84B1 has the highest activity toward IAA, with
significant activity also shown toward IBA, IPA, and cinnamic acid. The
other proteins only showed low activity toward these additional
substrates. The activity of UGT84B1 toward indole acetyl aspartate,
indole acetyl glutamate, indole acetyl alanine, and indole acetaldehyde
was also tested and found to be zero (data not shown). In addition to
UDPG, the ability of UDP-galactose and UDP-xylose to act as sugar
donors was investigated and found to be negative.
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Table I
Specific activity of UGT84B1, UGT84B2, UGT75B1, and UGT75B2 with
different substrates
All assays were carried out in 50 mM HEPES, pH 7.6, 2.5 mM MgSO4, 10 mM KCl, 1 mM
potential substrate, 5 mM UDPG, and 0.5-25 µg/ml enzyme.
The reactions were incubated at 30 °C for 20-60 min. UDP-galactose
and UDP-xylose were tested at 2.5 mM in the same
conditions. ND, not
determined.
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Effect of Potential Inhibitors and Activators of Enzyme
Activity--
The effect of metal ions on UGT activity is shown in
Table II. Some increase in relative
activity was observed with Mg2+, Ca2+, and
Mn2+, which was abolished by the addition of EGTA/EDTA.
Inclusion of UDP led to a decrease in activity, either arising from
product inhibition or from a shift in equilibrium of the reaction that is known to be reversible (30). No effects were observed with the
addition of either zeatin or dihydrozeatin.
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Table II
Effect of potential activators and inhibitors on the activity of
UGT84B1
Each assay contained 1 µg/ml UGT84B1, 50 mM HEPES, pH
7.6, 1 mM IAA, and 5 mM UDPG and was incubated
at 30 °C for 10 min. The relative activities with respect to
controls containing no additions were assigned to be 100%. Activities
were determined using HPLC. Results represent the average of three
independent replicates ± S.D.
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Kinetic Analysis of the Activity of UGT84B1--
The initial rate
of activity (millikatals kg
1) at differing
concentrations (mM) of both IAA and UDPG is plotted in Fig.
4. The results indicate that a ternary
complex is required for catalysis. Confirmation of this requirement for
both IAA and UDPG binding to the enzyme before catalysis was obtained
by results of incubations involving only UGT84B1 and UDPG, in which no
free UDP could be detected (data not shown). The inset shows
the secondary plots of these data. Table
III summarizes the kinetic analysis for
IAA and three other substrates: IBA, IPA, and cinnamic acid. The data show that, in vitro, IBA, IPA, and cinnamic acid are
substrates comparable with IAA. Competition experiments were used to
compare IAA with the other two naturally occurring substrates, IBA and cinnamic acid (Fig. 5). Conjugation of
IAA or IBA is barely affected by the presence of cinnamic acid. When
IAA and IBA compete with each other, IAA is found to be a better
competitor. This suggests that IAA is the preferred substrate when
compared with cinnamic acid and IBA.

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Fig. 4.
Kinetic analysis of UGT84B1. A
representative double reciprocal plot for UGT84B1 at 0-1
mM IAA and 0-5 mM UDPG was determined by
the coupled assay at 30 °C. The corresponding secondary plots are
shown in the inset.
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Table III
Kinetic parameters for UGT84B1
The coupled assay was used to determine the kinetic parameters of
UGT84B1. Each assay contained substrate at a range from 0 to 1 mM. A saturating concentration of UDPG at 5 mM
was used to measure the enzyme activity towards IBA, IPA, and cinnamic
acid. The results shown represent the mean of three independent
experiments ± S.D. ND, not determined.
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Fig. 5.
Competition assays. The relative
activity of UGT84B1 toward IAA, IBA, or cinnamic acid, compared with no
competitor, was determined by HPLC with competing substrates (0.1 or 1 mM), 5 mM UDPG, and 50 mM HEPES, pH
7.6, following incubation for 4 min at 30 °C. The reaction was
terminated by trichloroacetic acid. The values given are the average of
three individual replicates ± S.D.
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Potential Inhibition of UGT84B1 Activity by Phospholipids--
A
hydrophobicity plot of UGT84B1 indicated hydrophobic regions in the
protein sequence, suggesting a possible site(s) of interaction with
hydrophobic substrate(s) and/or membrane phospholipids. The effect of
phospholipids on UGT84B1 was investigated in two ways. First, a direct
effect of including phospholipids with differing charges
(phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine, and phosphatidic acid) on enzyme activity was
analyzed, but no change was observed (data not shown). Second, binding
of the UGT84B1 to phospholipids was investigated: again, no effect was
observed. As shown in Fig. 6, in contrast
to a tomato annexin (Rp35), a soluble protein known to bind to
phospholipids in a calcium-dependent interaction (32),
UGT84B1 was recovered in the soluble fraction under all conditions
assayed.

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Fig. 6.
Interaction of the UGT84B1 with
phospholipids. Phospholipid vesicles prepared from a mixture of
phosphatidylserine and phosphatidylcholine were incubated with tomato
annexin GST fusion (Rp35) or UGT84B1 in the presence (+) or absence
( ) of 1 mM Ca2+, 1 mM
Mg2+ in 50 mM HEPES, pH 7.6. Protein bound in a
Ca2+-dependent manner was separated from
nonbound protein by centrifugation and subsequently eluted with 5 mM EDTA. Samples were analyzed on a 10% SDS-PAGE gel, and
proteins were visualized by Coomassie staining.
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Expression of UGT84B1 in Arabidopsis Plants--
The sequence
UGT84B1 was identified in the genome data base as a putative
UGT on the basis of a UDP-glucose binding motif previously reported
(1). However, no expressed sequence tag had been found to correspond to
UGT84B1; therefore, there was no direct evidence to show the
gene is expressed in planta. Fig. 7 shows the results of analyzing
UGT84B1 mRNA levels. Clearly, the data, based on RT-PCR,
show that UGT84B1 transcripts are highly abundant in
siliques and inflorescence with relatively low levels of expression
observed in root tissue.

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Fig. 7.
RT-PCR analysis of different plant
tissues. The expression of UGT84B1 in different plant
tissues was analyzed by RT-PCR on 1% (w/v) agarose gel
(upper) and by Southern hybridization using radiolabeled
UGT84B1 DNA fragments following exposure of 10 s
(middle), or longer exposure of 1 h (lower).
The controls are samples prepared without reverse transcriptase.
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DISCUSSION |
The near completion of the Arabidopsis genome
sequencing project provides an opportunity to identify novel plant
genes on the basis of homology searching. In this study, we have used a characteristic signature motif, previously shown to define a family of
UGTs encompassing UDP-glucuronosyltransferases and
UDP-glucosyltransferases from prokaryotic and eukaryotic organisms (1).
A detailed phylogenetic analysis of the Arabidopsis
multigene family encoding the enzymes has shown that 12 groups (A-L)
can be distinguished on the basis of their sequence and pattern of
intron gain (2). In particular, a subfamily in group L has been
identified in biochemical analyses of recombinant proteins to form
glucose esters (27). These studies provided the basis for screening
Arabidopsis UGTs for their ability to form the glucose ester
conjugate of IAA. When the recombinant proteins were analyzed for their
ability to glucosylate IAA, only one, UGT84B1, showed high activity.
Interestingly, the closest relative of UGT84B1, with greater than 80%
identity, had only trace activity toward IAA. Six of these recombinant
proteins, including UGT84B1, are active toward cinnamates (27).
Previously, the only gene that has been identified to encode a UGT of
IAA is the maize iaglu (16). In that study, the gene was
found by screening an expression library with antibodies raised against
a maize UGT. Following heterologous expression of the gene in E. coli, the bacterial extract was shown to have UGT activity toward
IAA. The maize sequence has been used for many years to identify
putative IAA-UGT in the Arabidopsis genome and expressed sequence tag data bases. For example, based on their close sequence homology, UGT75D1 has been annotated as an IAA-UGT (34). However, analysis of recombinant protein from this sequence for activity toward
IAA has proved negative. The fact that UGT84B2 with greater than 80%
identity to UGT84B1 and existing within the same plant species has only
negligible activity toward IAA demonstrates that sequence comparison
alone cannot be used to predict substrate specificity.
This study shows that in the in vitro assay, the recombinant
enzyme from UGT84B1 conjugates IBA, IPA, and cinnamic acid
in addition to IAA. The Km values for IAA, IBA, IPA,
and cinnamic acid are nearly identical, although competition
experiments indicate that IAA is the preferred substrate for UGT84B1
when compared with cinnamic acid and IBA (Table III, Fig. 5). The
interaction of UGT84B1 and IAA is complex and can be affected by a
number of factors. The specificity studies indicate that the chain
length of the carboxyl group is important, since UGT84B1 has very low activity toward ICA. The enzyme activity is further affected by the
hydroxyl groups on the indole and benzyl rings. A 64% decrease in the
activity has been observed with 5-OH IAA compared with that with IAA. A
similar effect has been found with the cinnamates. Caffeic acid, which
has two hydroxyl groups on the benzyl ring, shows the lowest activity
among the cinnamates. In addition, the enzyme has extremely low
activity toward 2-oxIAA, an IAA derivative containing a carbonyl group
at position 2 on the indole ring (Table I, Fig. 3).
There are no previous data available on a purified IAA-UGT, since the
maize enzyme was analyzed only as a partial purified extract (38, 39)
and the recombinant product of the maize iaglu gene was
neither purified nor characterized (16). The properties of the
recombinant enzyme analyzed in this study are different from those
described for the partially purified IAA-UGT from maize, with respect
to the lack of inhibition by cytokinins and lack of effect of
phospholipids, whereas the dependence on metal ions and reducing
conditions are identical. Cinnamic acid, IPA, and IBA were not tested
as potential substrates of the maize enzyme. Similar levels of trace
activities for enzymes of the two species were observed toward ferulic
acid and p-coumaric acid.
This study is a biochemical characterization of a recombinant protein
using an in vitro assay. As yet, the relationships of this
activity to events that occur within the plant are unknown. For
example, both IBA and IAA glucose conjugates have been identified in
plants, including Arabidopsis (23, 40, 41). IAA is the preferred substrate for UGT84B1 in vitro, but in
planta the enzyme may glucosylate both IAA and IBA dependent on
cell specificity of expression, relative availability of substrate(s),
and relative compartmentalization of enzyme and substrate(s).
Preliminary analysis of the tissue-specific expression of
UGT84B1 in Arabidopsis plants showed the highest
level of expression in the siliques, when steady-state levels of
UGT84B1 mRNA were detectable even in Northern analysis
of total RNA. Results from RT-PCR, however, demonstrate transcripts of
the gene are also present in the root. Experiments are currently under
way using a UGT84B1 promoter-reporter gene system to define
more closely which cells express the UGT. Parallel phenotypic
characterization of transgenic Arabidopsis plants either
overexpressing UGT84B1 or with the gene knocked out by a
nonautonomous dSpm transposable element insertion will provide
essential information on the function of the IAA-UGT.