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Plant Physiol. (1999) 119: 173-178
An in Vitro System from Maize Seedlings for
Tryptophan-Independent Indole-3-Acetic Acid
Biosynthesis1
Anders Östin,
Neboj a Ili , and
Jerry D. Cohen*
Department of Forest Genetics and Plant Physiology, The Swedish
University of Agricultural Sciences, 90183 Umeå, Sweden (A.Ö.); Department of Plant Biology, University of Maryland, College Park,
Maryland 20742 (N.I.); and Horticultural Crops Quality Laboratory,
Beltsville Agricultural Research Center, Agricultural Research Service,
United States Department of Agriculture, Beltsville, Maryland
20705-2350 (J.D.C.)
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ABSTRACT |
The enzymatic synthesis of
indole-3-acetic acid (IAA) from indole by an in vitro preparation from
maize (Zea mays L.) that does not use tryptophan (Trp)
as an intermediate is described. Light-grown seedlings of normal maize
and the maize mutant orange pericarp were shown to
contain the necessary enzymes to convert [14C]indole to
IAA. The reaction was not inhibited by unlabeled Trp and neither
[14C]Trp nor [14C]serine substituted for
[14C]indole in this in vitro system. The reaction had a
pH optimum greater than 8.0, required a reducing environment, and had
an oxidation potential near that of ascorbate. The results obtained with this in vitro enzyme preparation provide strong, additional evidence for the presence of a Trp-independent IAA biosynthesis pathway
in plants.
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INTRODUCTION |
Until recently, the biosynthesis of IAA was hypothesized to occur
via oxidative decarboxylation and deamination of the amino acid Trp
(Bandurski et al., 1995 ). Trp is an important intermediate for IAA
biosynthesis in microorganisms and in plants (Patten and Glick, 1996;
Normanly, 1997). However, there is now strong genetic and in
vivo-labeling evidence for a Trp-independent IAA biosynthesis pathway
in several plant species (Baldi et al., 1991; Wright et al., 1991;
Michalczuk et al., 1992; Normanly et al., 1993). In some plants and/or
developmental stages, the Trp-independent pathway is the primary route
for IAA production. It appears to be of particular importance during
embryogenesis, when fine control over low levels of IAA are critical to
polar development (Michalczuk et al., 1992; Ribnicky, 1996). There have
been earlier reports of in vitro IAA biosynthesis with maize (Zea
mays L.) using extracts prepared from liquid endosperm tissue
(Jensen and Bandurski, 1994; Rekoslavskaya and Bandurski, 1994;
Rekoslavskaya, 1995) and coleoptiles (Koshiba and Matsuyama, 1993). The
liquid endosperm preparations from maize kernels converted indole to
IAA in vitro. This result suggested that the Trp-independent pathway
might contribute significantly to the newly synthesized IAA; however,
extensive Trp-to-IAA conversion also occurs in such preparations
(Rekoslavskaya, 1995; Ili et al., 1998). This finding is
consistent with our observation in carrot that tissues accumulating
relatively large quantities of IAA seem to use predominately Trp
conversion (Michalczuk et al., 1992) and that maize liquid endosperm is
a very active IAA-producing tissue (Jensen and Bandurski, 1994).
In another report, Trp-dependent IAA biosynthesis was detected in an in
vitro extract from maize coleoptiles (Koshiba and Matsuyama, 1993). IAA
biosynthesis was dependent on indole-3-acetaldehyde oxidase activity in
combination with the activity of a cytosolic ascorbate peroxidase. This
in vitro system used L- and D-Trp without any
discrimination and both L- and
D-aminotransferases were present in this tissue extract
(Koshiba and Matsuyama, 1993). In vivo-labeling studies using exogenous
[14C]Trp labeling of dark-grown coleoptile tips
showed metabolism of approximately 10% of the label into
[14C]IAA (Koshiba et al., 1995). This tissue is
competent to produce IAA from Trp but may do so only if the section is
removed from the rest of the plant and/or if a high level (18 µM) of Trp is used exogenously (Koshiba et al., 1995).
Overall, these results must be viewed in contrast with the compelling
evidence provided by 2H2O
and precursor labeling experiments. Such studies have shown that
coleoptiles of intact maize seedlings do not produce significant IAA
from de novo biosynthesis (Pengelly and Bandurski, 1983; Jensen and Bandurski, 1996), and [15N]Trp tracer
studies with another monocotyledonous plant have shown that
D-Trp is not an IAA precursor (Baldi et al.,
1991).
The maize mutant orange pericarp is a Trp auxotroph as a
result of mutations of both genes coding for Trp synthase  (Wright et al., 1992). Light-grown orange pericarp seedlings have
higher increased levels of total IAA, indicating that there is a
biosynthetic pathway to IAA that does not use Trp as an intermediate
(Wright et al., 1991). This was confirmed by experiments with
[15N]anthranilic acid or
2H2O labeling of
orange pericarp seedlings, which showed incorporation of the
label into IAA but not into Trp. In an effort to determine the
intermediates and isolate the enzymes involved in the Trp-independent IAA biosynthetic pathway in plants, we have established an in vitro
system in which the conversion of indole to IAA can be studied. Unlike
the genetic composition of endosperm (which is triploid, the product of
triple fusion), biochemical measurements from both normal and
orange pericarp genetic materials can be easily compared using preparations from light-grown maize seedlings.
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MATERIALS AND METHODS |
Reagents
All chemicals were from Sigma unless stated otherwise.
[2-14C]Indole (55 mCi/mmol),
[carboxyl-14C]IAA (57 mCi/mmol), and
L-[side chain-3-14C]Trp (53.8 mCi/mmol) were purchased from American Radiolabeled Chemicals (St.
Louis, MO). L-[U-14C]Ser (120 mCi/mmol) was purchased from Moravek Biochemicals (Brea, CA).
Plant Material
In most experiments, 8- to 9-d-old seedlings of maize (Zea
mays L. cv Silver Queen) (The Meyer Seed Co., Baltimore, MD) were used to prepare enzyme extracts. Maize kernels were soaked in running
tap water overnight and then planted in moist vermiculite. The plants
were grown in a growth room under constant light (cool-white fluorescent lamps, 30 µmol cm 2
s1) and at 25°C. Some studies used extracts
prepared from seedlings grown from kernels of the maize mutant
orange pericarp (Wright et al., 1991), and these kernels
were a gift from Dr. Allen Wright. Immature maize kernels were obtained
from mature Silver Queen plants (from the same seed lot that was used
for the seedling experiments) grown in a field plot during the summer
of 1996 (Beltsville, MD). Unpollinated ears were obtained by placing a
wax-paper bag over the ears before silk emergence to prevent
fertilization.
Preparation of the Maize Seedling IAA Biosynthesis in Vitro
System
The homogenization buffer consisted of 75 mM POPSO,
0.05 M
(NH4)2SO4,
0.25 M ascorbic acid, 10% (v/v) glycerol, 1.5% (w/v) PVP,
1% (w/v)
N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, 10 mM EGTA, 1 mM PHMB, 5 mM  -amino-n-caproic acid, 1 mM PMSF, 1 mM benzamide, 1 mM benzamidine, 50 µg/mL antipain, 1 mg/mL
pepstatin, and 50 µg/mL leupeptin in deionized glass-distilled water.
The homogenization buffer was prepared from a 2× stock of POPSO,
glycerol, (NH4)2SO4,
N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and PVP stored at 4°C. Just before preparation of the enzyme extract, the ascorbic acid was added and the pH adjusted to 8.5 with KOH. Finally, the protease inhibitors EGTA, PHMB, PMSF,
-amino-n-caproic acid, benzamide, benzamidine, antipain,
leupeptin, and pepstatin were added and the solution was diluted to its
final concentration. The homogenization buffer was chilled at  20°C
to a partially frozen slurry before use. These procedures were adapted
with modifications from the general recommendations of Gegenheimer
(1990) for preparation of plant enzyme extracts. Two modifications of
this basic buffer system were also used. To determine the optimum pH
for the complete reaction, an experiment was conducted in which the 75 mM POPSO buffer was changed to a mixed buffer
system composed of 40 mM POPSO plus 40 mM Mes. This mixed buffer allowed a pH range of 6.0 to 8.5 to be analyzed without changing the organic buffers present
in the reaction. In studies with maize orange pericarp seedlings, 75 mM Tris was substituted for 75 mM POPSO, but all other components remained the
same.
Maize seedlings were homogenized in the ice-cold homogenization buffer
slurry (10 g of seedlings/50 mL of buffer slurry) using a prechilled
mortar on salted ice in a 5°C cold room. The homogenized mixture was
then filtered through one layer of cheesecloth plus one layer of
Miracloth (Calbiochem). The pH was adjusted, if necessary, to 8.5 with
KOH. The extract was then centrifuged at 12,000g for 20 min
at 0°C to 5°C. The supernatant was decanted and used as the source
of enzymes, endogenous reactants, and cofactors for the in vitro
system. The amount of protein was determined by the Bradford dye method
(Bio-Rad).
Measurement of Enzyme Activity
One-milliliter aliquots of the plant extract were mixed with
200,000 dpm of [14C]indole or
[14C]Trp (1.7 µM) and incubated
for 5 h at 25°C or 35°C. In studies with seedling extracts
from the maize mutant orange pericarp 600,000 dpm of
[14C]indole or [14C]Trp
(5.1 µM) was used in each reaction. The
reaction was stopped by adjusting the pH to 2.7 with
H3PO4 and the solution was
partitioned against ethyl acetate. The ethyl acetate was held overnight
at 20°C to remove residual water by freezing. The sample was
centrifuged at 3000g for 45 s and the water droplet at
the bottom of the test tube was removed and discarded. The remaining
fraction was dried using a stream of nitrogen gas, resuspended in 20 µL of ethyl acetate, and 50 ng of unlabeled IAA was added as a
chromogenic tracer (Ehmann, 1977). The sample was applied to a Silica
Gel 60 TLC plate (20 × 20 cm, Merck, Darmstadt, Germany), which
was developed using solvent S1tlc
(chloroform:methanol:water, 85:14:1 [v/v]). The radioactivity on the
plate was detected using a radioimaging system (AMBIS 4000, Scanalytics, Inc., Billerica, MA). The IAA chromogenic standard was
visualized using Ehmann's reagent (Ehmann, 1977). The plate was dipped
briefly in the reagent, the color was developed by treatment for 5 min
at 100°C, and then the plate was washed under running water for 15 min to remove excess reagent. The Ehmann color reaction allowed IAA to
be visualized to compensate for changes in RF
caused by interfering compounds in the samples. Control samples were
boiled for 5 min before addition of radiolabeled indole or Trp.
Please note: (a) this TLC system has been characterized in terms of the
RF of a large number of indolic compounds, as
described by Sztein et al. (1995); (b) the radioactive compounds other
than indole shown in the boiled control were not present in the
[14C]indole before incubation with the boiled
enzyme sample; and (c) Trp remains in the initial water phase and does
not partition into the ethyl acetate phases applied to the TLC
plate.
In Vitro System for IAA Production from Maize Liquid-Endosperm
Extracts
Maize liquid-endosperm extracts (Ili et al., 1998) were
prepared essentially according to the methods of Rekoslavskaya and Bandurski (1994) and Rekoslavskaya (1995). Experiments were performed using maize liquid endosperm from both fertilized and unfertilized maize ears. To determine if the reaction was dependent on Trp as an
intermediate, 50 µg of different metabolites (Gly, IAA, indole,
indole-3-butyric acid, Ser, and L-Trp) was added to the reactions and the effects of these compounds on IAA formation were
determined.
GC-MS Analysis
Four standard incubations of seedling extracts with
[14C]indole, as described above, were pooled
and the IAA was isolated. In these four reactions, unlike those used
for TLC analysis, no carrier IAA was added. IAA was purified by ethyl
acetate partitioning, C18 HPLC on an UltraCarb
(30) column (4.6 × 50 mm, Phenomenex, Torrance, CA) using
27% methanol/water plus 1% acetic acid as the mobile phase, followed
by methylation with diazomethane (for additional details, see
Ribnicky, 1996). The sample was analyzed by GC-MS monitoring of
four ions in the selected-ion mode. Ions at m/z 130 (quinolinium ion) and m/z 189 (molecular ion) were monitored
for unlabeled methyl-IAA, and for
methyl-[14C]IAA the corresponding ions at
m/z 132 and 191 were monitored.
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RESULTS |
We have achieved the in vitro enzymatic biosynthesis of IAA in a
preparation from maize seedlings in which
[14C]indole was converted into
[14C]IAA (Fig. 1,
lanes C). This biosynthesis was independent of Trp because (a)
[14C]Trp was not converted to
[14C]IAA (Fig.
2); (b) the addition of unlabeled Trp did
not inhibit the [14C]indole conversion to
[14C]IAA (Fig. 1, lanes D); and (c) Ser did not
enhance the same conversion (data not shown). The addition of unlabeled
indole decreased the formation of [14C]IAA
(Fig. 1, lanes F). The addition of unlabeled IAA reduced the
[14C]IAA we observed (Fig. 1, lanes E)
and resulted in the accumulation of an unknown
14C-labeled compound. This metabolite was ethyl
acetate-soluble and had a RF slightly higher than
that of IAA. The formation of IAA in this reaction was verified by
GC-MS selected-ion monitoring analysis of its methylated derivative
(Fig. 3). The endogenous IAA can be
identified by its molecular ion at m/z 189 and its quinolinium ion at m/z 130, whereas the IAA formed from
[14C]indole was measured at +2 mass units, e.g.
m/z 191 and 132. As an additional confirmation of the Trp
independence of the reaction, IAA biosynthesis was measured in the in
vitro system from orange pericarp mutant seedlings (Fig.
4). This mutant lacks any functional Trp
synthase and thus cannot make Trp (Wright et al., 1991, 1992).
Thus, the results of this experiment provide further evidence excluding
the possibility that formation of IAA was via Trp.
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| Figure 1.
Silica-gel TLC analysis of the radiolabeled
reaction products obtained from [14C]indole. The reaction
products were partitioned at acidic pH into ethyl acetate and 50 ng of
unlabeled IAA was added before concentration and application to the TLC
plate. Except for the IAA standard and the boiled control, duplicate
reactions were spotted in adjacent lanes, as shown. After development
in solvent S1tlc, radiolabeled products were visualized
using an AMBIS 4000 radioimaging system. The RF values of
IAA and indole (as indicated on the abscissa) were confirmed by
visualization of the indole and the added unlabeled IAA using the
reagent of Ehmann (1977) and superimposing the two images. A potential
intermediate that accumulated with the addition of unlabeled IAA (lanes
D) and was not detected in the reactions with added unlabeled indole
(lanes F) is indicated on the abscissa (I?). Quantification of
radioactivity at the RF of IAA (obtained by software
analysis of the scanned image and correction for counting efficiency)
is shown in the bar graph at the top of the figure. The data on the
graph for C, D, E, and F are the averages of the values from each pair
of lanes shown.
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| Figure 2.
Results were obtained with the in vitro assay as
described for Figure 1, and compared with results obtained from maize
endosperm. Note that the endosperm preparations were able to
convert [14C]Trp as well as [14C]indole to
IAA, whereas only [14C]indole was an effective substrate
with the seedling preparations.
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| Figure 3.
Selected ion chromatogram (bottom) and selected
ion spectrum (top) for IAA formed in the enzyme reaction. The sample
was purified by C18 HPLC, methylated, and analyzed by
GC-MS. Ions at m/z 130 and 189 are characteristic of
unlabeled methyl-IAA resulting from unlabeled IAA or indole in the
reaction mixture from the plant enzyme preparation. Ions at
m/z 132 and 191 are the corresponding ions for the
methyl ester from the 14C-labeled IAA formed from the
radioactive indole supplied to the reaction.
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| Figure 4.
Results were obtained from in vitro preparations
of orange pericarp seedlings of maize. Conditions are as
described for Figure 1. A1 to A3 are three replicated reactions using
[14C]indole in the reaction mixture, and for replicates
C1 to C3 the radiolabeled substrate was [14C]Trp. Trp
does not partition into ethyl acetate from the aqueous phase, so it is
not shown on these chromatograms. Quantification of radioactivity
at the RF of IAA (obtained by software analysis of the
scanned image and correction for counting efficiency) is shown in the
bar-graph inset (data from A1-A3 and C1-C3 were averaged).
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Because of earlier reports of IAA biosynthesis in a maize
liquid-endosperm preparation (Rekoslavskaya and Bandurski, 1994; Rekoslavskaya, 1995), we compared the liquid endosperm in vitro system
with our seedling in vitro system (Fig. 2). The liquid endosperm
preparation was capable of converting
[14C]indole, [14C]Trp
(Fig. 2), and [14C]Ser (data not shown) to
[14C]IAA. In contrast, the maize seedling in
vitro system did not convert any detectable amounts of
[14C]Trp to [14C]IAA
(Fig. 2). Maize liquid endosperm prepared according to the seedling in
vitro protocol did not form any IAA from
[14C]indole or
[14C]Trp; similarly, maize seedlings prepared
according to the endosperm protocol produced no detectable
[14C]IAA from
[14C]indole or [14C]Trp
(data not shown).
The pH optimum for the reaction was found to be at or above pH 8.0 to
8.5 (Fig. 5). The temperature dependence
of the in vitro system showed 35°C to be better than 15°C and
25°C; additionally, although low levels of glycerol (10%) were
necessary, higher levels inhibited the reaction (Fig.
6). The addition of 50 µg of Gly, indole-3-butyric acid, or Ser to the reaction mixture had little or no
effect on the formation of [14C]IAA and, as
expected, [14C]Ser was not an effective
substrate for this reaction (data not shown).
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| Figure 5.
The effect of pH on the formation of IAA from
[14C]indole by in vitro preparations from maize
seedlings. For this experiment, the standard reaction was changed to a
mixed buffer system composed of 40 mM POPSO plus 40 mM Mes. This mixed buffer allowed a pH range of 6.0 to 8.5 to be analyzed without changing the organic buffers present in the
reaction.
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| Figure 6.
The effect of changes in the glycerol
concentration and incubation temperature on the amount of
[14C]IAA formed from [14C]indole by in
vitro preparations from maize seedlings during a 5-h incubation.
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DISCUSSION |
Trp has long been considered to be the sole or primary precursor
of the plant hormone IAA (Normanly et al., 1995), and Trp-dependent IAA
biosynthesis pathways have been characterized in several plant systems.
At least three pathways have been described from different plants
(Cohen and Bialek, 1984). Recently, evidence has accumulated for an
alternative pathway for the biosynthesis of IAA that does not use Trp
as an intermediate, and this pathway has now been shown to be
the primary route for IAA production in several plant species (Wright
et al., 1991; Michalczuk et al., 1992; Normanly et al., 1993, 1995;
Bartel, 1997). In trying to develop a useful in vitro system to study
Trp-independent IAA biosynthesis, our initial interest focused on the
maize liquid-endosperm preparations because they were simple to
prepare, were stable to freezing, and had high activity for IAA
biosynthesis. Evidence that this system probably included both
Trp-dependent and -independent IAA biosynthesis has been reported by
Rekoslavskaya and Bandurski (1994). However, both our own results (Fig.
2; Ili et al., 1998) and data presented by Rekoslavskaya (1995)
show that the majority of IAA was made from Trp by liquid-endosperm
preparations. Specifically, with the liquid-endosperm extracts we
observed that the formation of [14C]IAA from
[14C]indole was enhanced by the addition of Ser
and inhibited by the addition of unlabeled Trp (Ili et al.,
1998). This was true of endosperm preparations from both fertilized
(Fig. 2) and unfertilized (data not shown) ears. Because of the high
level of Trp conversion to IAA by endosperm preparations, it was not
possible to measure significant Trp-independent indole conversion in
such extracts. Therefore, we changed our focus to maize seedling
extracts, because results from experiments with the orange
pericarp mutant (Wright et al., 1991) have shown that this tissue
used only the Trp-independent pathway for biosynthesis of IAA.
Plant leaves are known to have an indole-oxidizing system (Nair and
Vaidyanathan, 1964; Chauhan et al., 1978), which often makes in vivo
use of applied indole unsuccessful. Thus, Wright et al. (1991) were
able to label maize seedlings and follow IAA biosynthesis successfully
with 2H2O and anthranilate,
but not with indole. However, in our in vitro system these oxidizing
enzymes are apparently inhibited at pH 8.5 (pH 5.0 is the optimum for
the maize indole-oxidizing activity [Chauhan et al., 1978]) and by
the use of PHMB, which is also known to inhibit such enzymes (Nair and
Vaidyanathan, 1964). The pH optimum for IAA formation (Fig. 5)
indicates that the maximal activity for the rate-limiting step would
occur in an alkaline cell compartment, such as that found in plastids. However, we cannot exclude the possibility that the pH optima data are
in part a reflection of optima of competing reactions for the same
substrate. We also found that the redox potential in the reaction
mixture had to be carefully controlled. Initial studies using 5 mM DTT or dithioerythritol gave variable results, which
were dependent on the individual enzyme preparation. Somewhat better
results were obtained when diethyldithiocarbamate was also included.
When ascorbate was used as a "redox buffer," consistently higher
yields of IAA were obtained. Thus, the reactions from indole to IAA
involve at least one step that is sensitive to the oxidation/reduction potential of the reaction environment.
In vitro IAA biosynthesis in extracts of young maize seedlings involves
the direct conversion of [14C]indole to
[14C]IAA without Trp as an intermediate. This
was verified by using preparations from orange pericarp
mutant seedlings. Because orange pericarp seedlings lack a
functional Trp synthase , in these extracts no Trp could be formed
from the supplied indole. Furthermore, the addition of unlabeled Trp
did not alter the conversion of [14C]indole to
[14C]IAA, whereas the addition of unlabeled IAA
resulted in the accumulation of a potential intermediate. These
results, together with the results from the maize liquid-endosperm
preparations, show that there are two different pathways of IAA
formation in maize and, as has been shown for carrot (Michalczuk et
al., 1992), that the expression of these two pathways is a function of
the plant's developmental stage. Further studies on the regulation and
activation of these pathways in plants will be important for
understanding hormonal, metabolic, and molecular regulation of plant
development. Experiments in our laboratories are currently directed
toward isolation of the intermediates in this process to understand the metabolic route for Trp-independent IAA biosynthesis.
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FOOTNOTES |
1
This study was supported by the U.S. Department
of Energy (grant no. DE-AI02-94ER20153) and by the Swedish Council for
Forestry and Agricultural Research.
*
Corresponding author; e-mail jdcohen{at}wam.umd.edu; fax
1-301-504-5107.
Received July 1, 1998;
accepted September 26, 1998.
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ABBREVIATIONS |
Abbreviations:
PHMB, p-hydroxymercuribenzoic
acid.
POPSO, piperazine-N,N -bis(2-hydroxypropanesulfonic
acid).
| |
ACKNOWLEDGMENTS |
We thank Dr. Janet P. Slovin (U.S. Department of
Agriculture-Agricultural Research Service Climate Stress Laboratory,
Beltsville, MD) and Dr. Jennifer Normanly (Department of Biochemistry
and Molecular Biology, University of Massachusetts, Amherst) for their comments on the manuscript. Support for A.Ö. by the Swedish
Council for Forestry and Agricultural Research is gratefully
acknowledged.
| |
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