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Plant Physiol. (1998) 118: 285-296
Metabolism of Indole-3-Acetic Acid in Arabidopsis1
Anders Östin,
Mariusz Kowalyczk,
Rishikesh P. Bhalerao, and
Göran Sandberg*
Department of Forest Genetics and Plant Physiology, The Swedish
University of Agricultural Sciences, S-901 83 Umeå, Sweden
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ABSTRACT |
The metabolism of indole-3-acetic
acid (IAA) was investigated in 14-d-old Arabidopsis plants grown in
liquid culture. After ruling out metabolites formed as an effect of
nonsterile conditions, high-level feeding, and spontaneous
interconversions, a simple metabolic pattern emerged. Oxindole-3-acetic
acid (OxIAA), OxIAA conjugated to a hexose moiety via the carboxyl
group, and the conjugates indole-3-acetyl aspartic acid (IAAsp) and
indole-3-acetyl glutamate (IAGlu) were identified by mass spectrometry
as primary products of IAA fed to the plants. Refeeding experiments
demonstrated that none of these conjugates could be hydrolyzed back to
IAA to any measurable extent at this developmental stage. IAAsp was further oxidized, especially when high levels of IAA were fed into the
system, yielding OxIAAsp and OH-IAAsp. This contrasted with the
metabolic fate of IAGlu, since that conjugate was not further
metabolized. At IAA concentrations below 0.5 µM, most of
the supplied IAA was metabolized via the OxIAA pathway, whereas only a
minor portion was conjugated. However, increasing the IAA concentrations to 5 µM drastically altered the metabolic
pattern, with marked induction of conjugation to IAAsp and IAGlu. This investigation used concentrations for feeding experiments that were
near endogenous levels, showing that the metabolic pathways controlling
the IAA pool size in Arabidopsis are limited and, therefore, make good
targets for mutant screens provided that precautions are taken to avoid
inducing artificial metabolism.
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INTRODUCTION |
The plant hormone IAA is an important signal molecule in the
regulation of plant development. Its central role as a growth regulator
makes it necessary for the plant to have mechanisms that strictly
control its concentration. The hormone is believed to be active
primarily as the free acid, and endogenous levels are controlled in
vivo by processes such as synthesis, oxidation, and conjugation. IAA
has been shown to form conjugates with sugars, amino acids, and small
peptides. Conjugates are believed to be involved in IAA transport, in
the storage of IAA for subsequent use, in the homeostatic control of
the pool of the free hormone, and as a first step in the catabolic
pathways (Cohen and Bandurski, 1978 ; Nowacki and Bandurski, 1980;
Tuominen et al., 1994; Östin et al., 1995; Normanly, 1997). It is
generally accepted that in some species conjugated IAA is the major
source of free IAA during the initial stages of seed germination (Ueda
and Bandurski, 1969; Sandberg et al., 1987; Bialek and Cohen, 1989),
and there is also evidence that in some plants (but not all; see Bialek
et al., 1992), the young seedling is entirely dependent on the release of free IAA from conjugated pools until the plant itself is capable of
de novo synthesis (Epstein et al., 1980; Sandberg et al., 1987).
The function of conjugated IAA during vegetative growth is somewhat
less clear. It has been shown that conjugated IAA constitutes as much
as 90% of the total IAA in the plant during vegetative growth
(Normanly, 1997). However, the role of the IAA conjugates at this stage
of the plant's life cycle remains unknown. Analysis of endogenous IAA
conjugates in vegetative tissues has revealed the presence of a variety
of different compounds, including indole-3-acetyl-inositol, indole-3-acetyl-Ala, IAAsp, and IAGlu (Anderson and Sandberg, 1982;
Cohen and Baldi, 1983; Chisnell, 1984; Cohen and Ernstsen, 1991;
Östin et al., 1992). Studies of vegetative tissues have indicated
that IAAsp, one of the major conjugates in many plants, is the first
intermediate in an irreversible deactivation pathway (Tsurumi and Wada,
1986; Tuominen et al., 1994; Östin, 1995). Another mechanism that
is believed to be involved in the homeostatic control of the IAA pool
is catabolism by direct oxidation of IAA to OxIAA, which has been shown
to occur in several plant species (Reinecke and Bandurski, 1983;
Ernstsen et al., 1987).
One area in the study of IAA metabolism in which our knowledge is
increasing is the analysis of the homeostatic controls of IAA levels in
plants. It has been possible, for instance, to increase the levels of
IAA in transgenic plants expressing iaaM and iaaH genes from Agrobacterium tumefaciens. Analysis of these
transgenic plants has indicated that plants have several pathways that
can compensate for the increased production of IAA (Klee et al., 1987; Sitbon, 1992). It is expected that future studies using now-available genes will provide further insight into IAA metabolism. For example, a
gene in maize encoding IAA-Glc synthetase has been identified, and
several genes (including ILR1, which may be involved
in hydrolysis of the indole-3-acetyl-Leu conjugate) have been cloned
from Arabidopsis (Szerszen et al., 1994; Bartel and Fink,
1995). Furthermore, Chou et al. (1996) identified a gene that
hydrolyzes the conjugate IAAsp to free IAA in the bacterium
Enterobacter aggloremans.
Because of its small genome size, rapid life cycle, and the ease of
obtaining mutants, Arabidopsis is increasingly used as a
genetic model system to investigate various aspects of plant growth and
development. IAA signal transduction is also being investigated
intensively in Arabidopsis in many laboratories (Leyser, 1997). Mutants
with altered responses to externally added auxins or IAA conjugates
have been identified in Arabidopsis. The identified mutants are either
signal transduction mutants such as axr1-4 (Lincoln et al.,
1990), or have mutations in genes involved in auxin uptake or
transport, such as aux1 and pin1 (Okada et al., 1991; Bennett et al., 1996). A few mutants that are unable to regulate
IAA levels or are unable to hydrolyze IAA conjugates, sur1-2
and ilr1, respectively, have also been identified (Bartel and Fink, 1995; Boerjan et al., 1995). To our knowledge, no mutant that
is auxotrophic for IAA has been identified to date, which may
reflect the redundancy in IAA biosynthetic pathways or the lethality of
such mutants.
In spite of the work reported thus far, many aspects of the metabolism
of IAA in Arabidopsis require further investigation, because few
details of the processes involved in IAA regulation are known. This
lack of knowledge puts severe constraints on genetic analysis of IAA
metabolism in Arabidopsis. For example, it is essential to have prior
knowledge of IAA metabolism to devise novel and relevant screens with
which to identify mutants of IAA metabolism. We have sought to address
this issue by identifying the metabolic pathways involved in catabolism
and conjugation under conditions that minimally perturb physiological
processes. In this investigation we studied the conjugation and
catabolic pattern of IAA by supplying relatively low levels of labeled
IAA and identifying the catabolites and conjugates by MS. Different feeding systems were tested to optimize the application of IAA and to
avoid irregularities in metabolism attributable to culturing, feeding
conditions, or microbial activity. It is well documented that IAA
metabolism is altered according to the amount of exogenous auxin
applied; therefore, we placed special emphasis on distinguishing between catabolic routes that occur at near-physiological
concentrations and those that occur at the high auxin concentrations
commonly used in mutant screens.
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MATERIALS AND METHODS |
Chemicals and Isotopically Labeled Substrates
[1 -14C]IAA, with a specific activity of
55 mCi/mmol, was purchased from American Radiolabeled Chemicals (St.
Louis, MO). [13C6]IAA was
from Cambridge Isotope Laboratories (Andover, MA). All other chemicals
were from Sigma if not stated otherwise. Labeled IAAsp, IAAGlu, and
OxIAA were synthesized from [1-14C]IAA
according to the methods of Tuominen et al. (1995) and Ili et
al. (1997). To obtain metabolic profiles, a solution of 50% Murashige-Skoog medium (Duchefa, Haarlem, The Netherlands) with 5 µM radiolabeled [1-14C]IAA
metabolite was used. For the identification of metabolites, a 10 µM 1:1 mixture of nonlabeled
IAA:[13C6]IAA spiked with
1 µM [1-14C]IAA as a tracer in
one-half-strength Murashige-Skoog medium was used. For feeds with low
concentrations of IAA, 0.1 and 0.5 M solutions of
[5-3H]IAA were used (specific activity of 20 Ci/mmol, American Radiolabeled Chemicals).
Plant Material
Seeds of Arabidopsis ecotype Columbia were surface sterilized in
5% calcium hypochlorite plus 0.02% (v/v) Triton X-100 for 30 min, and
then washed three times with sterile water. They were then dried
overnight and stored at 4°C until use. Seeds grown in soil were
planted without any surface sterilization.
Feeding Conditions
Plantlets were potted and grown under short-day conditions until
they had about 20 leaves. They were then picked from the soil, the
roots were rinsed with distilled water, and the root system was removed
under water approximately 5 mm from the lowest leaf. The shoots were
transferred to a tissue-culture dish with 200 µL of medium containing
labeled IAA in each well. After the labeling solution was taken up by
the transpiration stream, excess 50% Murashige-Skoog medium was added
to provide the plant with enough liquid for the rest of the incubation
period. Plants were also grown in Petri dishes containing
Murashige-Skoog medium, 0.3% agar, and 3% Suc. After 14 d in
short-day conditions, the plantlets were carefully rinsed with sterile
water, transferred to 24-well plates (Costar 24, Cambridge, MA), and
fed with 5 µM radiolabeled
[1-14C]IAA in full-strength Murashige-Skoog
medium, pH 5.6, in the dark. Finally, 20 surface-sterilized seeds were
transferred to a 250-mL conical flask containing 50 mL of
Murashige-Skoog medium, pH 5.6, with 3% Suc. Germination and further
growth took place in long-day conditions for 14 d, during which
time the medium was replaced once. The plants were then fed with
radiolabeled [1-14C]IAA,
[1-14C]IAAsp,
[1-14C]IAGlu, or
[1-14C]OxIAA (at approximately 5 µM) in the dark. In separate experiments, 0.5 and 0.1 µM radiolabeled [5-3H]IAA were
fed to replicate plants in the dark.
To assess the effects of OxIAA, IAA, IAGlu, and IAAsp, Arabidopsis
seeds were germinated on Murashige-Skoog medium with 3% Suc or on
Murashige-Skoog medium with 3% Suc supplemented with these compounds
at a concentration of 50 µM. After 4°C treatment of the
seeds in the dark for 2 d, the plates were placed in continuous light. The effect of the compounds on root growth was assessed 10 d after germination.
Extraction and Purification
The plant material was homogenized in liquid nitrogen using a cold
mortar and pestle. The homogenized material was then extracted with
methanol containing 0.02% (w/v) diethylcarbamatic acid as an
antioxidant for 5 h. Before reduction to the aqueous phase in a
rotary evaporator, 10 mL of distilled water was added to the filtered
extraction mixture. The aqueous phase was then adjusted to pH 2.7, and
the sample was passed through a C18 solid-phase extraction column (Varian, Harbor City, CA) and eluted with 80% methanol. The aqueous phase of the samples used for metabolite identification was partitioned three times against ethyl acetate, after
adjustment to pH 2.7, and then partitioned three times against water-saturated butanol. Each of the three phases, ethyl acetate, butanol, and aqueous, was dried and redissolved in methanol, diluted with 1% acetic acid, and applied to C18
solid-phase extraction columns as described above. The eluates from the
solid-phase extraction were reduced in volume and centrifuged in 1.7-mL
microcentrifuge tubes at 20,800g for 5 min before
HPLC.
HPLC
Metabolic profiles were obtained, and the metabolites were
purified by HPLC before MS analysis. The HPLC system consisted of a
controller and pump (type 600, Waters, Millipore), and samples were
introduced by an autosampler onto a 3.9- × 150-mm, 5-µm ODS symmetry
column (Waters, Millipore). The mobile phase was delivered at a flow
rate of 0.8 mL/min with an initial mixture of 5% methanol in water
(with 1% acetic acid in both solvents) for 5 min, followed by a 45-min
linear gradient to 60% methanol, and finally, a linear gradient to
90% methanol over 5 min. For analysis of
14C-labeled samples, the eluate was passed
through a radioactivity monitor (model 9701, Reeve Analytical Ltd.,
Glasgow, Scotland) with a heterogeneous flow cell packed with
cerium-activated lithium glass scintillate. For analysis of
3H samples, the heterogenous flow cell was
replaced with a 500-µL flow cell and operated in homogenous mode. The
scintillant was Permablend III (Packard Instrument Co., Meriden, CT)
used in a 4:1 (v/v) scintillant:eluate ratio. One-minute fractions were collected (fraction collector, ISCO, Lincoln, NE) for further MS
analysis. Overall control, data sampling, and data handling were
performed by the Millennium data system (Millipore). Aliquots of the
extracts were mixed with [14C]OxIAA,
[14C]IAAsp, or
[14C]IAGlu before injection into the HPLC
system to identify peaks comigrating with these potential metabolites.
Hydrolysis Experiments
Aliquots from HPLC-purified metabolites were stirred in 1 M NaOH for 1 h at room temperature (25°C). Under
these conditions, ester (but not amide) conjugates of IAA are
hydrolyzed (Bandurski and Schulze, 1974). At the end of the incubation
period, samples were neutralized with glacial acetic acid and purified
on C18 solid-phase extraction columns, then
analyzed by HPLC as described above. Metabolites were also treated with
7 M NaOH at 100°C for 3 h, under a stream of
water-saturated nitrogen gas, which results in the hydrolysis of amide
IAA conjugates (Bandurski and Schulze, 1974). After strong alkaline
hydrolysis, samples were cooled on ice for 15 min, adjusted to pH 3.0 with phosphoric acid, and processed in the same way as the samples from
weak alkaline hydrolysis.
Derivatization
To improve chromatography and sensitivity in the HPLC-MS analysis
(Östin, 1995), free carboxylic acids were methylated in HPLC-purified fractions according to the method of Shlenk and Gellerman (1960). After methylation, the samples were reseparated by
HPLC as described above. Radioactive fractions were then analyzed by
HPLC-MS. Aliquots of the methylated radioactive fractions were purified
a second time by HPLC, and were then dried, dissolved in 25 µL of
acetonitrile, and silylated (before further analysis by GC-MS) by
adding 25 µL of bis(trimethylsilyl)-trifluoroacetamide with 1% (v/v)
trimethylchlorosilane (Pierce) and heating in sealed vials at 70°C
for 15 min. Finally, they were reduced to dryness, redissolved in
acetonitrile, and analyzed by GC-MS.
Analysis by GC-MS and HPLC-MS
GC-MS was performed using a gas chromatograph (model 5890, Hewlett-Packard) linked to a mass spectrometer (model JMS-SX-102, JEOL). Samples were injected, splitless, at 280°C onto a 15-m × 0.25-mm fused-silica column with low-bleed MS film (Sil-8 CB, ChromPack, Middelburg, The Netherlands), 0.25 µm thick. The carrier gas was helium. The column temperature was programmed to increase 20°C/min from 70°C to 280°C, and was held at final temperature until elution of the sample. Ions were generated with 70 eV at an
ionization current of 300 µA. The acceleration voltage was 10 kV.
Positive-ion mass spectra were obtained at a rate of 1 s per scan
for a mass range of m/z 25 to 800. The capillary
HPLC-frit-FAB-MS system used for the identification of the
metabolites has been described in detail elsewhere (Östin et al.,
1992). For the initial HPLC separation, a 300- × 0.32-mm capillary
column packed with 5-µm ODS symmetry packing material was used (LC
Packings, Amsterdam, The Netherlands). The ion source temperature was
50°C, and ions were generated with a beam of 5 to 6 kV xenon atoms at
an emission current of 20 mA. The acceleration voltage was 10 kV.
Positive-ion mass spectra were obtained at a rate of 5 s per scan
for a mass range of m/z 50 to 800, and the
spectra were background subtracted. All MS data were processed by a
data system (MD7000, JEOL).
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RESULTS |
Incubation Systems
IAA metabolism was investigated in three different systems:
soil-grown, tissue-cultured, and liquid-cultured Arabidopsis plants. Figure 1 shows the metabolic profile
after feeding with 5 µM
[1-14C]IAA. In all three systems, five major
metabolites, numbered 1 through 5, were always found. In the soil-grown
plants and in plants fed with high levels of IAA, additional polar
metabolites could be detected in some experiments. Some of these
additional peaks were attributed to the nonsterile conditions and some
to spontaneous conversions. In this investigation we chose to perform the incubation in darkness to minimize light-induced oxidation of IAA.
Plants grown in sterile conditions were chosen for the rest of the
investigations, partly to avoid complications arising from metabolism
by contaminating microorganisms and partly to allow rapid and
reproducible uptake of the label. The system with plants grown in
liquid culture proved to be efficient for producing large amounts of
metabolites, whereas incubation of single, sterile-grown plantlets in
wells allowed specific labeling via the Arabidopsis transpiration
stream.
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| Figure 1.
Gradient-elution reversed-phase HPLC-radioactivity
chromatogram of partially purified extracts of Arabidopsis
plants fed [1-14C]IAA. All incubations were done in
darkness.
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Identification of IAA Metabolites
To facilitate identification of the unknown metabolites,
Arabidopsis plants grown in the dark in liquid culture were fed a 1:1
mixture of nonlabeled IAA and
[13C6]IAA spiked with
[1-14C]IAA. The total IAA concentration was 5 µM. Using an equal amount of unlabeled and labeled
([13C6]IAA) gives
characteristic fragments with two ions of equal size separated by six
mass units. The metabolites denoted 1 through 5, plus four additional
polar metabolites denoted 6 through 9, were purified as
described in ``Materials and Methods''. Fractions were collected
after HPLC purification and processed for MS analysis as described
below. The use of ion-suppression reversed-phase separation gives the possibility of calculating retention times relative to IAA, thus giving
an indirect estimate of relative polarity at the pH of the analysis.
Metabolite 1
Metabolite 1 had a retention time relative to IAA of 0.74. When
subjected to either mild or strong hydrolysis, only decomposition occurred, with no release of IAA, indicating that the compound was not
an IAA conjugate. No co-elution with known standards occurred when
aliquots of the extract were spiked with labeled OxIAA, IAGlu, or
IAAsp. Methylation with diazomethane altered some of this compound to a
species with an identical retention time to that of metabolite 5. The
underivatized metabolite was subjected to capillary
HPLC-frit-FAB-MS analysis, producing the spectrum shown in
Figure 2A, and GC/MS analysis of
the silylated compound gave a spectrum as in Figure 2B. The
HPLC-MS spectrum, corresponding to that of OxIAA linked to a hexose
sugar, consists of a protonated molecular ion [MH]+ at
m/z 354/360. Loss of the sugar moiety
gives OxIAA, as indicated by the fragment at
m/z 192/198.
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| Figure 2.
Frit-FAB-MS spectra of metabolite 1 (A);
and GC-MS spectrum of silylated metabolite 1 (B) and of the silylated
hydrolysis product of metabolite 1 (C).
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The OxIAA moiety is then further fragmented to
m/z 174/180 and
m/z 146/152. Silylation of the catabolite
before GC-MS caused significant hydrolysis to free OxIAA, identified by
analysis of its trimethylsilyl derivative (Fig. 2C). The silylated
derivative of metabolite 1 yielded at least two isomers, with spectra
showing a molecular ion at m/z 785/791,
corresponding to the incorporation of six trimethylsilyl groups into an
OxIAA-hexose conjugate. The m/z 450, 361, 217, 204, 191, 147, and 73 fragmentation pattern has been demonstrated
previously using
1-O-(indole-3-acetyl)- -D-glucopyranose (Ehman, 1974) to represent a silylated sugar moiety.
Furthermore, the ions m/z 290 and 407 represent a shift of +88 mass units, corresponding to a silylated
oxygen group in the number 2 position of an oxindole. These peaks also
show the diagnostic double labeling from
[13C6]IAA (plus 6 mass units), with fragments
at m/z 296 and 413. We thus conclude that
metabolite 1 is OxIAA linked to a hexose sugar via the carboxyl group.
We did not proceed with a detailed characterization of the sugar
because this was not the main focus of the study.
Metabolite 2
This metabolite had a retention time of 0.81 relative to IAA. Both
mild and strong hydrolysis caused breakdown of the metabolite without
any release of IAA. Spiking experiments demonstrated co-elution with [14C]OxIAA (Fig.
3). Methylation with diazomethane changed
the retention time to the same as that of metabolite 5. Analysis with
capillary HPLC-frit-FAB-MS of the methylated derivative revealed
a spectrum corresponding to an OxIAA methyl ester (Table
I), and GC-MS of the methylated and
silylated derivative revealed a spectrum corresponding to that of a
di-(trimethylsilyl)-OxIAA methyl ester (Table
II). We concluded, therefore, that
metabolite 2 is OxIAA.
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| Figure 3.
Time-course study of a [1-14C]IAA
feed to Arabidopsis plants grown in liquid culture for 2 to 120 h
and of partially purified extracts spiked with [14C]OxIAA
and [14C]IAAsp.
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Table I.
Tabulated FAB spectra of methylated IAA metabolites
The intensity of nonlabeled peaks are shown in parentheses, the
intensity of the 13C6 label from the IAA feed
is of approximately equal intensity as that of nonlabeled ions. Bp,
Base peak with relative intensity 100%. All spectra also contain an
adduct ion [M + glycerol + H]+ with an
intensity of 2% to 10%; these are not tabulated.
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Table II.
Tabulated EI spectra of methylated/silylated IAA
metabolites
The intensity of the nonlabeled peaks are shown in parentheses, the
intensity of the 13C6 label from the IAA feed
is of approximately equal intensity as that of nonlabeled ions. Bp,
Base peak with a relative intensity of 100%.
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Metabolite 3
Metabolite 3 had a retention time of 0.87 relative to IAA.
Hydrolysis experiments resulted in the release of IAA with strong but
not with mild hydrolysis, indicating that the compound is an amide
conjugate of IAA. Spiking experiments resulted in co-elution with
[14C]IAAsp (Fig. 3). Methylation with
diazomethane changed the retention time of metabolite 3 to a relative
retention of 1.06, corresponding to the methylation of the two
carboxylic acids in the Asp moiety (as demonstrated by standard IAAsp
having the same shift in retention time after methylation). The
structure of IAAsp was verified by comparing the HPLC-MS spectrum of
its methylated derivative and the GC-MS spectrum of its
methylated/silylated derivative with appropriate standards, as shown in
Tables I and II.
Metabolite 4
Metabolite 4 had a retention time of 0.90 relative to IAA.
Hydrolysis experiments indicated that the compound was an amide conjugate of IAA. Spiking experiments resulted in co-elution with [14C]IAGlu (data not shown). Methylation with
diazomethane changed the retention time of metabolite 4 to a relative
retention of 1.23, corresponding to the methylation of the two
carboxylic acids in the Glu moiety. The structure of IAGlu was verified
by analyzing the HPLC-MS spectrum of its methylated derivative and the
GC-MS spectrum of its methylated/silylated derivative (Tables I and II).
Metabolite 5
In samples from all feeding systems, a metabolite with more or
less the same retention time as IAA (0.98 relative to IAA) on
ion-suppression chromatography was found. This compound was also formed
from metabolite 1 during sample preparation and methylation with
diazomethane. Methylated metabolite 2 has the same retention time in
HPLC. After derivatization and subsequent GC-MS analysis, this peak was
identified as a di-(trimethylsilyl)-OxIAA methyl ester. We believe that
this metabolite is a result of spontaneous methylation of metabolite 1, a phenomenon we have earlier observed for
IAA-1-O--D-Glc during storage in acid
methanol. Therefore, we excluded this metabolite from the following
discussion of physiologically relevant metabolites.
Metabolites 6 to 9
In soil-grown plants and in sterile-grown plants given high
dosages of IAA, additional oxidative IAAsp metabolites were detected as
their methylated derivatives by HPLC-MS. Metabolites 6 to 9 have
molecular ions that correspond to an extra oxygen incorporated into
IAAsp ([M + H]+ m/z
335/341 with [13C6]
label). These metabolites had a relative retention to IAA of 0.21, 0.31, 0.41, and 0.47, respectively, on a 3.9- × 150-mm, 5-µm ODS
column (Nova Pac, Waters, Millipore), using the same solvent system as
described above. Metabolites 8 and 9 were major constituents that
showed the same fragmentation pattern as the standard OxIAAsp methyl
ester and the same retention time as the two spontaneously formed
stereoisomers (Table I). Compared with methylated OxIAAsp, metabolites
6 and 7 lacked the fragment m/z 204 (which
corresponds to loss of the entire methylated side chain) and have in
addition two pairs of ions, m/z 130/136 and
132/138, which probably result from the loss of oxygen from the
m/z 146/152 quinolonium fragment. These spectra
are characteristic of hydroxylated forms of IAAsp (Table I).
Furthermore, some other minor metabolites with the characteristic
12C6/13C6
label were detected from plants given high dosages of IAA, but these
were not fully characterized because of their questionable physiological relevance. OxIAAsp is most likely metabolized to a
metabolite with a molecular ion m/z 592/598. Two
compounds with the expected
12C6/13C6
ratio in their molecular ion (m/z 365/371 and
379/385) were detected (data not shown). An unknown ester-bound
conjugate of IAA with a molecular ion of m/z
372/378 was also detected (data not shown). Methylated OxIAAsp can be
partially oxidized to a hydroxylated OxIAAsp during isolation or when
stored in solution for longer times (data not shown).
Time-Course Studies
In a pulse-chase experiment 5 µM
[1-14C]IAA was fed to liquid-culture-grown
plants that were harvested at various times from 2 to 120 h after
treatment. The resulting metabolic profiles are illustrated in Figure
3. OxIAA is formed early and is subsequently conjugated to form an
OxIAA hexose. Amide conjugation is seen initially by measuring the
formation of IAGlu and, at a later stage, IAAsp. IAGlu accumulated over
time and was not further metabolized, whereas IAAsp accumulated as long
as there was free IAA available. IAAsp was further metabolized
to the polar catabolites described above. It is also worth noting that
accumulation of more stable forms occurs mainly through the formation
of OxIAA hexose in older plants and IAAsp in younger plants (data not
shown).
Metabolism of OxIAA, IAAsp, and IAGlu
An important question is whether the major metabolites formed
after feeding plants with labeled IAA are strictly catabolites or if
they can be reused (i.e. converted back to IAA). This was investigated
in two separate experiments. [14C]OxIAA,
[14C]IAAsp, and
[14C]IAGlu were separately fed to
liquid-culture-grown Arabidopsis plants. Feeding with IAAsp indicated
that this conjugate was stable in Arabidopsis tissues, with almost no
metabolism occurring during the first 24 h. However, after 48 h there was significant metabolism, with both a minor level of
hydrolysis to IAA and formation of additional catabolites of IAAsp
(Fig. 4A). The results of feeding with
labeled IAGlu are presented in Figure 4B. IAGlu was stable over time,
with only minor levels of metabolism occurring. This is consistent with
the time-course studies of IAA metabolism shown in Figure 3, which show
that IAGlu accumulated over time and did not appear to be metabolized
further. Finally, Figure 4C shows that OxIAA was, as expected, not
converted back to IAA, and that the major product of supplied OxIAA was
OxIAA hexose (peak 1). There was also a polar peak that co-eluted with
DiOxIAAsp (metabolite 10) and was probably formed via OxIAAsp.
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| Figure 4.
Time-course study of [1-14C]IAAsp
(A), [1-14C]IAGlu (B), and [1-14C]OxIAA (C)
feeds to Arabidopsis plants grown in liquid culture for 6, 12, and
48 h.
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In a second experiment the sensitivity of Arabidopsis seedlings to the
major catabolites/conjugates OxIAA, IAAsp, and IAGlu was tested using a
protocol described earlier for IAAsp (Bartel and Fink, 1995). OxIAA,
IAAsp, and IAGlu had no effect at all on the phenotype after the
feedings, even at very high concentrations (data not shown). This,
together with the data from the feeding experiments, clearly shows that
these three catabolites/conjugates cannot be reconverted back to IAA at
measurable rates in Arabidopsis at this developmental stage.
Physiological Relevance of the Metabolic Studies
Many studies involving metabolic analysis of IAA, especially
studies using Arabidopsis, have used relatively high concentrations of
IAA during the feeding of plants. To determine the consequence of
feeding level, we investigated the effect of the concentration of IAA
fed to the Arabidopsis on the metabolites detected. Metabolic profiles
were obtained from plants fed with 5, 0.5, and 0.1 µM IAA. Figure 5 shows a time-course study
with 0.5 µM IAA fed to the plants. This experiment
demonstrates that different metabolic profiles were obtained by feeding
with 0.5 and 5 µM IAA. Low-level feeding with IAA
resulted in the detection of mostly OxIAA and OxIAA hexose, with a
minor level of conjugation to IAAsp and IAGlu. A set of polar
catabolites was also detected, which most likely represented polar
products of OxIAA and IAAsp. The 3H profiles
presented in Figure 5 were analyzed using an on-line radioactivity
monitor operated in the homogenous mode. The signal-to-noise ratio is
significantly improved using this detection method compared with the
use of a heterogeneous detection system. This is demonstrated by the
resolution of the clustered peaks at approximately 13 min in Figure 5
(homogenous detection) compared with the same unresolved peaks in
Figure 3 (heterogeneous detection). Another interesting observation was
that the [5-3H]IAA low-level feed indicated a
more rapid turnover of IAA compared with the
[1-14C]IAA feed. When 0.1 µM IAA
was fed to the plants, the same general metabolism as with a 0.5 µM concentration (Fig. 5) was found (data not shown).
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| Figure 5.
Gradient-elution reversed-phase HPLC-radioactivity
chromatogram of aliquots of partially purified extracts of
sterile-grown plants fed [5-3H]IAA in liquid culture in
darkness for 6 to 48 h.
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DISCUSSION |
We analyzed the metabolism of IAA by Arabidopsis using three
different experimental systems. Irrespective of the feeding system, four metabolites of IAA were consistently detected. The metabolites were identified as the conjugates IAAsp and IAGlu, the oxidative catabolite OxIAA, and an OxIAA-hexose conjugate. Some additional metabolites, such as oxidative products of IAAsp, were found in profiles from older soil-grown material and from plants that were fed
very high levels of auxin. Metabolic studies with synthetically made
[14C]OxIAA, [14C]IAGlu,
or [14C]IAAsp indicated that free IAA was not
released from these conjugates in measurable amounts by Arabidopsis at
this developmental stage. Irreversible oxidation occurred mainly via
OxIAA, but the possibility that IAAsp can also function as an initial
catabolic intermediate is indicated by its further metabolism to
OxIAAsp and OH-IAAsp. In the soil-grown plants, relatively more
oxidative catabolites were formed, with OxIAA hexose accumulating over
time, whereas in the sterile-grown, young plant material, IAAsp and
IAGlu were the major end products.
Earlier results based on TLC separation indicated that IAA
applied to Arabidopsis was metabolized to compounds with an
RF similar to that of IAAsp and/or IAAGlu, IAGlc,
and indole-3-acetyl-Leu and/or indole-3-acetyl-Ile (Sztein et al.,
1995). Our results differ from the earlier observations made by Sztein
et al. (1995) in that we did not find any peaks that comigrated with
indole-3-acetyl-Leu, indole-3-acetyl-Ile, or IAGlc. Because IAGlc might
be spontaneously converted to IAA methyl ester during methanolic
extraction, we also attempted extraction with 80% acetone. This
extraction resulted in reduced spontaneous conversion of OxIAA hexose
to OxIAA methyl ester, but also in increased spontaneous degradation of
OxIAA and reduced recovery of the amide. No IAGlc was detected in
either case. Based on our results (see below), we believe that IAGlc is
a product that is detectable only when high levels of IAA are fed to
plant tissues. The overall function of conjugation to Glc needs to be
investigated further. For example, Catala et al. (1992) observed
that the only metabolite of IAA in tomato in which formation was not
inhibited by cyclohexamide was IAGlc. This, together with our data,
indicates that the induction of this pathway is complex.
Our strategy in this study was to use
[1-14C]IAA, first to establish the metabolic
profiles, and second, to produce larger amounts of catabolites for
identification. The results from [1-14C]IAA
feeding were then complemented by results from
[5-3H]IAA feeding, which were used to support
the physiological relevance of the identified metabolites. The average
values for endogenous concentration of IAA in the Arabidopsis plants
used for these studies varied between 10 and 25 ng/g fresh weight,
depending on the growth conditions and developmental stage. In a
separate investigation, we found by GC-MS microanalysis that the
concentration range within individual Arabidopsis plants ranges from 5 to more than 900 ng/g (K. Ljung, R. Bhalerao, and G. Sandberg,
unpublished data), the higher values being found in actively growing
parts of the plant.
The concentration of IAA conjugates in the Arabidopsis plants was very
high, with average values for the amide conjugates reaching micrograms
per gram fresh weight levels. We used an average of 5 µM
IAA solution, added in 5-fold excess (v/w), in our initial feeding
experiments and this would be expected to cause changes in the
endogenous IAA pool. However, because the values in different organs
ranged from 5 to 900 ng/g, the endogenous pool in the old, nonexpanding
leaves was increased a maximum of 175-fold, whereas the pool in the
most rapidly expanding leaves could be increased less than 2-fold by
such an application. The metabolic profiles were therefore reanalyzed
using [5-3H]IAA at a concentration of 0.5 µM. In this case the dilution of the average pool could
be expected to increase 5-fold, whereas the dilution of the pool in the
nonactively growing parts would be 17.5-fold, and that in actively
dividing parts, less than 4%. The concentration used may alter the
endogenous pools markedly in some of the plant compartments, but not in
others. We decided, therefore, to further reduce the concentration of
IAA fed to the plants. A new feed was performed with 0.1 µM [5-3H]IAA. In this case, the
dilutions of the IAA pools were 1-fold when calculated at the
whole-plant level, 3-fold in the old leaves, and less than 1% in the
actively growing parts. These estimates are based on the fact that no
active accumulation occurred, so at most a 5-fold increase is
theoretically possible.
As shown in Figure 5, OxIAA and OxIAA hexose are the major
catabolites when low levels of IAA are fed to the plant. Thus, at IAA
concentrations less than 0.5 µM, the majority of the
metabolism is via OxIAA, and only a minor portion of IAA is conjugated.
However, at 5 µM concentrations of IAA, the metabolic
pattern is altered (Figs. 1 and 3), with a marked induction of
conjugation to IAAsp and IAGlu. The conjugative pathways to IAAsp and
IAGlu may be relevant to normal physiological processes, because the
actively growing parts of the Arabidopsis plant do contain high levels of IAA. Thus, the addition of 5 µM exogenous IAA may not
cause totally unphysiological conditions in these specific tissues. This aspect of tissue-specific effects has to be evaluated in future
experiments in which the metabolism in each tissue is related to the
actual endogenous concentration.
We can now conclude that the metabolism of IAA in Arabidopsis plants at
the developmental stage studied follows the pathway presented in Figure
6. These results also demonstrate
that nonsterile conditions or feeding high concentrations of IAA will
induce alternative metabolic pathways. Additionally, the complexities
of such analysis are increased by the inherent instability of the
indolic compounds, since they are both light sensitive and easily
oxidized. Therefore, data obtained using high IAA levels and nonsterile
conditions should be treated with caution, because they are unlikely to
reflect any normal physiological process in the plant. Until this
report, extremely high auxin dosages have been used in experiments with Arabidopsis aimed at analyzing metabolism of IAA and isolating mutants.
One exception is the study of IAA biosynthesis in Arabidopsis by
Normanly et al. (1993), in which low levels of IAA precursors were fed.
In our investigation, one of the major metabolites identified was OxIAA
hexose, which, during isolation, can be spontaneously methylated to
form OxIAA methyl ester, a compound that comigrates almost precisely
with IAA. Therefore, if identification and analysis are not done
carefully, this metabolite could easily be mistaken for free IAA
released from a conjugated form upon hydrolysis. Feeds with OxIAA and
hydroxylated forms of IAAsp also show that there are a number of polar
catabolites of both compounds that can also be formed by spontaneous
oxidation and that should not be confused with genuine in planta
catabolites. Thus, the results presented here are of consequence not
only for determining the metabolites of IAA produced by Arabidopsis,
but also for providing important information necessary for future
designs of screens aimed at the isolation of mutants in metabolic
pathways for IAA.
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| Figure 6.
Proposed metabolic pathway for IAA in Arabidopsis
plants. The solid arrows represent the steps that have been
demonstrated by in planta conversions under physiologically relevant
conditions.
|
|
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FOOTNOTES |
1
This work was financed by the Swedish Foundation
for Strategic Research and the European Commission DG XII biotechnology
program.
*
Corresponding author; e-mail goran.sandberg{at}genfys.slu.se; fax
46-90-786-5901.
Received March 25, 1998;
accepted June 15, 1998.
Abbrevations: FAB, fast-atom bombardment; IAAsp,
indole-3-acetyl Asp; IAGlc, indole-3-acetyl Glc; IAGlu,
indole-3-acetyl Glu; OxIAA, oxindole-3-acetic acid.
| |
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Top
Abstract
Introduction