Campanella et al, Plant Physiology, 2004

Campanella et al, Plant Physiology, 2004

A Novel Auxin Conjugate Hydrolase from Wheat
with Substrate Specificity for Longer Side-Chain
Auxin Amide Conjugates1
James J. Campanella*, Adebanke F. Olajide, Volker Magnus, and Jutta Ludwig-Müller
Department of Biology and Molecular Biology, Montclair State University, Montclair, New Jersey 07043
(J.J.C., A.F.O.); Rudjer Boskovic Institute, 10002 Zagreb, Croatia (V.M.); and Institut für Botanik,
Technische Universität Dresden, 01062 Dresden, Germany (J.L.-M.)
This study investigates how the ILR1-like indole acetic acid (IAA) amidohydrolase family of genes has functionally evolved in
the monocotyledonous species wheat (Triticum aestivum). An ortholog for the Arabidopsis IAR3 auxin amidohydrolase gene
has been isolated from wheat (TaIAR3). The TaIAR3 protein hydrolyzes negligible levels of IAA-Ala and no other IAA amino
acid conjugates tested, unlike its ortholog IAR3. Instead, TaIAR3 has low specificity for the ester conjugates IAA-Glc and IAAmyoinositol and high specificity for the conjugates of indole-3-butyric acid (IBA-Ala and IBA-Gly) and indole-3-propionic-acid
(IPA-Ala) so far tested. TaIAR3 did not convert the methyl esters of the IBA conjugates with Ala and Gly. IBA and IBA
conjugates were detected in wheat seedlings by gas chromatography-mass spectrometry, where the conjugate of IBA with Ala
may serve as a natural substrate for this enzyme. Endogenous IPA and IPA conjugates were not detected in the seedlings.
Additionally, crude protein extracts of wheat seedlings possess auxin amidohydrolase activity. Temporal expression studies of
TaIAR3 indicate that the transcript is initially expressed at day 1 after germination. Expression decreases through days 2, 5, 10,
15, and 20. Spatial expression studies found similar levels of expression throughout all wheat tissues examined.
In vascular plants, auxins, primarily indole-3-acetic
acid (IAA), regulate gene expression, cell division, and
cell elongation and differentiation in plant tissue.
Auxins also affect vascularization, phototropism, geotropism, fruit development, flower development, and
apical dominance (Davies, 1995). While IAA in low concentrations stimulates growth and development, higher
concentrations can be toxic to the plant (Bandurski
et al., 1995). Therefore, tight control of IAA concentration is necessary for proper plant development.
IAA is stored in conjugated forms that are mostly
considered to be inactive. Two main types of conjugated molecules have been studied: the amide-linked
IAA forms bound to one or more amino acids and the
ester-linked forms primarily bound to a sugar(s).
These two types of conjugates appear to be found at
varying concentrations in the diverse tissues of angiosperms (Domagalski et al., 1987). On average, 95% of
all IAA in a plant is conjugated into these storage
forms (Cohen and Bandurski, 1982; Bandurski et al.,
1995; Campanella et al., 1996; Walz et al., 2002).
There have been a variety of amide conjugates
found in the plants studied to date. IAA-Asp has
been identified as a natural conjugate in Scots pine
1
This work was supported in part by a Sokol grant for undergraduate research and in part by the Croatian Ministry of Science
and Technology (grant no. 0098080).
* Corresponding author; e-mail james.campanella@montclair.
edu; fax 419–791–9834.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.104.043398.
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(Pinus sylvestris; Andersson and Sandberg, 1982) and,
together with IAA-Glu, in cucumber (Sonner and
Purves, 1985) and soybean (Cohen, 1982). IAA-Ala
has been detected in Picea abies Karst (Östin et al.,
1992). Additionally, IAA-Ala, IAA-Asp, IAA-Leu, and
IAA-Glu have been detected in Arabidopsis L. Heynh
(Tam et al., 2000; Kowalczyk and Sandberg, 2001),
although recent data suggest that IAA peptides may
account for the majority of amide conjugates in this
and other plant species (Walz et al., 2002; Park et al.,
2003).
Common IAA-ester conjugates include IAAmyoinositol glycosides, IAA-myoinositol, and IAAGlc (Bandurski et al., 1969; Kopcewicz et al., 1974;
Bandurski and Schulze, 1977; Domagalski et al.,
1987). These ester conjugates have been identified in
many species over the last 60 years (Bandurski et al.,
1969; Slovin et al., 1999).
The ILR1-like IAA amidohydrolase gene family is
thought to be involved in the regulation of free IAA
concentrations. This gene family was originally characterized in the model plant Arabidopsis (Bartel and
Fink, 1995; Davies et al., 1999; LeClere et al., 2002). The
ILL1, ILL2, and IAR3 enzymes cleave IAA-Ala preferentially, while ILR1 prefers IAA-Leu and IAA-Phe as
substrates. Another ILR1-like family member, sILR1,
whose gene product has substrate specificity for IAAGly and IAA-Ala, has been isolated from the related
species Arabidopsis suecica Norrl. ex O.E. Schulz
(Campanella et al., 2003a, 2003d). Amidohydrolase
homologs have also been isolated from Brassica rapa
(Schuller and Ludwig-Müller, 2002). Additionally,
Plant Physiology, August 2004, Vol. 135, pp. 2230–2240, www.plantphysiol.org Ó 2004 American Society of Plant Biologists
Isolation of an Auxin Amidohydrolase from Wheat
several IAR3-like orthologs (MtIAR32, MtIAR33,
MtIAR34, and MtIAR35) have been cloned from the
non-Brassicaceae species Medicago truncatula Gaertn.
(J.J. Campanella, J. Ludwig-Müller, and S. Wexler,
unpublished data).
Since many of the IAA conjugates in monocots are
described as ester conjugates (Cohen and Bandurski,
1982), we hypothesized that IAA conjugate hydrolases
isolated from a grass might primarily be ester conjugate hydrolases. The first ester conjugate hydrolase
activity was found in maize (Hall and Bandurski,
1986). This work was followed by whole-seedling
studies examining movement and hydrolysis of IAAinositol (Chisnell and Bandurski, 1988). More recently,
Kowalczyk et al. (2003) identified an ester conjugate
hydrolase in maize, but the corresponding gene has
yet to be cloned. Therefore, our goal became the
identification and isolation of a putative IAA conjugate hydrolase from a major model monocot.
Our phylogenomic studies identified in wheat (Triticum aestivum) a full-length ortholog of the Arabidopsis IAA amidohydrolase IAR3 (Campanella et al.,
2003c). We employed this sequence data to clone the
full-length putative wheat hydrolase (TaIAR3) and
characterize it. Our goals were to better understand
how the functional molecular evolution of this important gene family has diverged between dicots and
monocots, and which auxin conjugates play a specific
role in monocots.
Upon expression and enzymatic characterization of
the TaIAR3 enzyme, we found that the enzymatic regulation of auxin in wheat may be more intricate than
we anticipated. TaIAR3 is an amidohydrolase that
preferably hydrolyzes amino acid conjugates of the
long-side-chain auxins indole butyric acid (IBA)
and indole propionic acid (IPA). This article reports
the first functional characterization of an amidohydrolase able to recognize these long-side-chain conjugate
species.
RESULTS
Substrate Specificity of TaIAR3
The DNA and protein sequences of TaIAR3 show
higher homology to the Arabidopsis ortholog IAR3
with 49% and 79% identity, respectively, than to any
other member of the ILR1-like family. A phylogenetic
analysis comparing the TaIAR3 protein against all the
members of the ILR1-like family confirms that IAR3
and TaIAR3 are most similar (Fig. 1). Both the IAR3
and TaIAR3 products contain putative endoplasmic
reticulum localization signals (data not shown), a hallmark of many of the auxin amidohydrolases (Davies
et al., 1999; LeClere et al., 2002; Campanella et al.,
2003a, 2003c).
After cloning the complete cDNA of TaIAR3 (sequence obtained from the The Institute of Genomic
Research [TIGR] Expressed Sequence Tag Index) into
Plant Physiol. Vol. 135, 2004
Figure 1. Neighbor-joining phylogram of Arabidopsis ILR1-like protein
family members orthologous to TaIAR3. The wheat hydrolase (bold)
demonstrates the closest homology to the IAR3 protein sequence
(bold); the high bootstrap value at the node of the tree (bold) indicates
a probability score of approximately 98% for the validity of this prediction. The scale at the bottom of the figure indicates relative genetic
distance.
pETBlue-2, the enzyme activity was determined in
vitro (Table I; Fig. 2). Enzyme activity from cells
containing an empty vector was compared with that
from cells containing the peTaIAR3 vector. These cells
were either incubated with Glc because the promoter
from pETBlue-2 is leaky, or induced with isopropyl
b–D-thiogalactopyranoside (IPTG). Relative expression levels of the TaIAR3 protein in Escherichia coli
were determined by direct comparison of induced and
uninduced bacterial proteins on SDS-PAGE (data not
shown). Densitometry analysis indicated a 2.7-fold
increase in expression of TaIAR3 in the induced cells
over background expression.
The level of enzyme activity was calculated after
comparing auxin release in uninduced and IPTGinduced cells. We observed a novel substrate specificity for longer side-chain auxins, especially IBA-Ala. In
addition, the amino acid attached to the auxin moiety
was also important, as shown by comparing IBA-Ala
and IBA-Gly as substrates. Methylation of their carboxyl groups abolished recognition by the enzyme
almost completely. Interestingly, the Ala conjugate of
an auxin with a side chain shorter by one methylene
group (IPA) was also accepted as a good substrate.
Further shortening of the side chain of the indole
derivative (IAA) almost completely abolished enzyme
activity.
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Campanella et al.
Table I. Enzyme activity of TaIAR3 expressed in E. coli
For each assay, approximately 2.5 mg total E. coli protein was used.
Mean values of three to five independent experiments 6SE are given.
The values for which no SE is given have been measured only once. The
activity is expressed as IAA formed in cells induced by IPTG, subtracted
by values from noninduced cells.
Substrate
Enzyme Activity
nmol auxin released
min21 mg21 protein
IAA-Asp
IAA-Ala
IAA-Gly
IAA-Leu
IAA-Ile
IAA-Phe
IAA-Val
IAA-Glc
IAA-inositol
IPA-Ala
IBA-Ala
IBA-Gly
Me-IBA-Ala
Me-IBA-Gly
0.04 6 0.03
0.02 6 0.01
0.03
0.12
0.06
0
0
0.5 6 0.1
0.2 6 0.04
8.8 6 2.2
14.8 6 3.6
4.9 6 1.5
0
0.8
IBA-Ala were used. IAA-Ala was negligibly hydrolyzed in 3-d-old seedlings at approximately 20% of the
enzyme activity toward the IBA-Ala substrate (Table
II). As seedlings aged to 6, 15, and 17 d, no IAA-Ala
hydrolase activity was detectable. IBA-Ala hydrolyzing enzyme activity was detected in all developmental stages investigated, being highest in 15- and
17-d-old seedlings. However, the analysis of whole
seedlings could mask localized concentration changes
in these compounds and in the hydrolysis activity.
Activity was specifically observed in cells after
induction with IPTG as shown for IBA-Ala as substrate (Fig. 2). Cells containing an empty vector as well
as uninduced cells showed only a very small peak at
the retention time (Rt) of IBA. This demonstrates the
specificity of the reaction. There were no other metabolites detectable (data not shown).
The reaction product obtained after enzymatic hydrolysis of IBA-Ala with the Rt of IBA was collected
from HPLC, subsequently methylated and analyzed
by gas chromatography-mass spectrometry (GC-MS).
The methylated putative IBA showed the same Rt as
a methylated IBA standard in the ion chromatogram,
and the mass spectrum showed the characteristic ions
of the molecular ion at m/z 217 and the quinolinium
ion at m/z 130 (data not shown).
Other potential substrates for TaIAR3, such as IBA
and IPA conjugates with Asp, will be investigated in
the future.
Determination of Free and Conjugated IAA and IBA
and Amidohydrolase Activity in Wheat Seedlings
To test whether the endogenous auxin content would
reflect the expression pattern of TaIAR3, the endogenous content of free and total IAA and IBA was determined during seedling growth (Fig. 3). While total
IAA and IBA decreased as expected during the first few
days after germination (IAA more than IBA), the
concentration of free IAA did not change significantly;
however, free IBA was significantly enhanced in 17-dold seedlings compared to 6-d-old seedlings, as determined by ANOVA (95% confidence). This is reflected
in the amidohydrolase activity measured in vitro in
extracts of seedlings when the substrates IAA-Ala and
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Figure 2. HPLC separation of the substrate, IBA-Ala, and the reaction
product, IBA, after incubation of the substrate with E. coli cell lysate for
1 h. Chromatograms were recorded at 280 nm. A, Standards of IBA and
IBA-Ala. B, Lysate of cells containing an empty vector. C, Lysate of cells
containing the peTaIAR3 vector uninduced. D, Lysate of cells containing the peTaIAR3 vector induced with IPTG.
Plant Physiol. Vol. 135, 2004
Isolation of an Auxin Amidohydrolase from Wheat
both young and mature plants (Fig. 4B). There is
a slightly higher expression level of the transcript seen
in coleoptiles during early development, and the same
is seen in adult stem tissues. Presumably, the TaIAR3
gene product is required to help stimulate early growth
of the coleoptile and continues to be of regulatory
importance throughout the life of the seedling and
into adulthood. Expression of the 18S control showed
no difference between samples examined (data not
shown).
DISCUSSION
Figure 3. Free and total IAA and IBA concentrations in wheat during
seedling development. The values presented are means 6 SE of three
independent experiments.
Identification of IBA-Ala in Wheat Seedlings
Extracts of wheat seedlings, as well as TaIAR3
in vitro, hydrolyzed IBA-Ala to a large extent. We
therefore analyzed extracts from wheat seedlings for
IBA-Ala as an endogenous compound. Following
treatment with diazomethane, a peak was found in
the ion chromatogram with the Rt of IBA-Ala methyl
ester (data not shown). This peak showed a mass
spectrum that is in accordance with the presence of
IBA-Ala in wheat seedlings. The molecular ion at
m/z 289, as well as the quinolinium ion at m/z 130, was
present, although the spectrum contained several
impurities. The latter are due to the problem that only
very small amounts of IBA-Ala were present.
Under the same conditions, no peak corresponding
to IPA-Ala was detectable in wheat extracts, indicating
that either very low concentrations are present or IPAAla is not a native substance in wheat seedlings.
Auxin conjugates play a major role in the regulation
of auxin homeostasis. While, for two Arabidopsis
species, conjugate hydrolases have been identified
that can hydrolyze amino acid conjugates of IAA
(LeClere et al., 2002; Campanella et al., 2003a), we
describe here the activity of a novel auxin conjugate
hydrolase activity from a monocot species (T. aestivum)
toward amino acid conjugates with IBA.
The isolation of an amidohydrolase from wheat
became possible with the availability of the sequenced
T. aestivum cDNAs from TIGR database, providing the
opportunity to identify novel plant genes on the basis of
homology searching. In this study, the sequence of the
Arabidopsis IAR3 gene was employed to identify an
orthologous gene (TaIAR3) in the wheat genome. It was
found that the TaIAR3 protein, although it has high
amino acid sequence homology to its Arabidopsis
ortholog, is able to cleave conjugates of auxin molecules
with longer side chains, IBA and IPA, while all other
auxin conjugate hydrolases so far identified cleaved
amino acid conjugates with IAA (LeClere et al., 2002;
Campanella et al., 2003a). In addition, the TaIAR3
mRNA shows a different spatial and temporal regulation compared to IAR3 from Arabidopsis (Davies et al.,
1999; Rampey et al., 2003), suggesting that the IAR3 and
TaIAR3 enzymes may be employed in different developmental pathways in Arabidopsis and wheat.
IBA is a naturally occurring auxin commonly used
to induce rooting (Hartmann et al., 1990). Several
studies have demonstrated within the last decade that
IBA is present in vascular plants (Epstein et al., 1989;
Ludwig-Müller and Epstein, 1991, 1993; Epstein and
Temporal and Spatial Expression of TaIAR3
The TaIAR3 transcript is first detectable in seedlings
at day 1 after germination and appears to fall steadily
in concentration until day 20 (Fig. 4A). It may be that
the high expression of the enzyme is required for
growth very early in plant development, but the
expression is down-regulated after that significant
phase of growth has passed. Expression of the 18S
control showed no difference between samples examined (data not shown).
Spatial expression of the TaIAR3 transcript appears
fairly homogeneous throughout the wheat tissues in
Plant Physiol. Vol. 135, 2004
Table II. Auxin conjugate hydrolase activity from wheat seedlings
during development using IAA-Ala and IBA-Ala as substrates
Mean values of three different enzyme preparations 6SE are given.
Enzyme Activity
Age of Seedlings
IBA-Ala
days after germination
0
3
6
15
17
IAA-Ala
pM auxin released min21 mg21 protein
1.0
1.4
3.7
1.8
0
6
6
6
6
0.1
0.1
0.3
0.2
0
0.4 6 0.06
0
0
0
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Campanella et al.
Figure 4. Temporal (A) and spatial (B) expression of TaIAR3 transcripts.
Graphs were generated using the relative standard curve method
(Applied Biosystems, 2001). A, Calibrator was the 1-d-old plant
expression data. B, Calibrator was the coleoptile expression data.
Ludwig-Müller, 1993; Ludwig-Müller et al., 1993). IBA
appears to be stored like IAA in a conjugated form
with amino acids or sugars (Epstein and LudwigMüller, 1993; Ludwig-Müller et al., 1993), allowing its
slow hydrolysis and release. Moreover, IBA may be an
endogenous precursor to IAA (Epstein and Lavee,
1984; Nordström et al., 1991; van der Krieken et al.,
1992; Baraldi et al., 1993). Conversely, IAA can be
converted to IBA in several species, including maize
and Arabidopsis (Epstein and Ludwig-Müller, 1993;
Ludwig-Müller and Hilgenberg, 1995; Ludwig-Müller
et al., 1995a; Ludwig-Müller, 2000).
IBA has been mostly disregarded in plant physiology
studies until recently because there were questions of
2234
whether or not it was a natural plant hormone. Several
studies suggest that IBA is important during the rooting process (Nordström et al., 1991; van der Krieken
et al., 1992). Ludwig-Müller et al. (1993) found that up
to 30% of auxin in Arabidopsis may be IBA. Some
experimental evidence suggests that IBA may act as an
endogenous growth hormone on both roots and stems
without conversion to IAA (Ludwig-Müller et al.,
1995b, 1995c; Chhun et al., 2003), although the recent
analysis of IBA-resistant Arabidopsis mutants revealed
that at least some of the auxin activity of IBA results
from its conversion to IAA by peroxisomal b-oxidation
(Zolman et al., 2000, 2001; Zolman and Bartel, 2004).
IPA was first found in decomposing organic matter,
likely as a Trp metabolite of microorganisms participating in the decay process. Bacteria can indeed produce IPA (Mohammed et al., 2003), but this capability is
limited to certain species (Elsden et al., 1976), while the
compound is inhibitory to others (Matsuda et al., 1998;
Walker et al., 2003). Some microbes have acquired the
ability to detoxify IPA by conjugation. Bacillus megatherium, for instance, couples the acid to Glc, Ala, Ser,
and Thr (Tabone and Tabone, 1953; Tabone, 1958). Seed
plants appear to take advantage of the sensitivity of
some bacteria to IPA to suppress pathogens; the compound was secreted by the roots of axenically grown
Arabidopsis treated with salicylic acid, a compound
that is usually formed in response to microbial attack
(Walker et al., 2003). Further research will show if the
occurrence of IPA in (nonsterile) Cucurbita pepo hypocotyls (Segal and Wightman, 1982) and in the roots of
Pisum sativum seedlings (Schneider et al., 1985) and the
preliminary evidence for its presence in Brassica oleracea
(Linser et al., 1954), Nicotiana tabacum leaves (Bayer,
1969), and Lycopersicon esculentum Mill cotyledons
(Aung, 1972) should be viewed as a response to
bacterial attack, as a result of bacterial contamination,
or as evidence for a role in plant development.
Exogenous IPA is somewhat less active than IAA in
stem-elongation assays (Fawcett et al., 1960; Katekar,
1979), but more active in the promotion of lateral
root growth on the primary root of pea seedlings
(Wightman et al., 1980). Conjugated forms of IPA have,
to our knowledge, not been identified in seed plants,
but it is likely that their roots are consistently exposed
to these compounds as metabolites of soil bacteria.
Three factors suggest that, in wheat, IBA-amide
conjugates are more likely to be the endogenous target
of the enzyme. First, we have identified the presence of
IBA and IBA conjugates in wheat seedlings, where the
enzymatic synthesis of IBA has also been demonstrated (Ludwig-Müller et al., 1995c), but we have
been unable to detect IPA conjugates. The very presence of IBA conjugates strongly supports the hypothesis that they are in vivo substrates. Second, although
TaIAR3 is able to cleave IPA-Ala conjugates efficiently,
it has a higher activity for IBA-Ala. Third, IBA, but not
IPA, synthesis was shown to occur in maize and IBA
synthesis was also demonstrated in wheat (LudwigMüller and Epstein, 1991; Ludwig-Müller et al., 1995c).
Plant Physiol. Vol. 135, 2004
Isolation of an Auxin Amidohydrolase from Wheat
Although the enzymatic hydrolysis of IBA conjugates has not been previously described in the literature, it is unclear whether this activity is unique to
monocots. Hydrolases from other plant species, such
as A. suecica (sILR1; Campanella et al., 2003d) and
M. truncatula (MtIAR32; J. J. Campanella, J. LudwigMüller, and S. Wexler, unpublished data) tested so far
have little or no activity against IBA-Ala and others
have not been reported.
Monocots diverged from dicots approximately 200
to 250 million years ago (Wolfe et al., 1989; Troitsky
et al., 1991; Kellogg, 2001). Therefore, IAR3 must
predate this point of divergence, since it is conserved
not only in the angiosperms but in the gymnosperms
as well (Campanella et al., 2003c). The gymnospermangiosperm divergence took place 300 to 360 million
years ago, suggesting that the ILR1-like family is quite
ancient and that its role is intimately tied to growth
and development in vascular plants (Troitsky et al.,
1991; Oliviusson et al., 2001; Albert et al., 2002; Cooke
et al., 2002, 2004).
From the experimental evidence provided so far, it
seems that different strategies to regulate auxin content
have evolved. While in charophytes and liverworts the
auxin conjugation rates are very slow and therefore
biosynthesis is a major contributor to free auxin, the
conjugation rate increases from hornworts and mosses
to vascular plants and thus also its importance in auxin
homeostasis (Cooke et al., 2002, 2004).
With the characterization of the TaIAR3 gene product, we are faced with additional questions of how
monocots and dicots differ in their regulation of auxin
homeostasis. Mapelli et al. (1995) reported that the
primary conjugates in wheat appear to be the IAAester type; however, the TaIAR3 enzyme appears to
have little to do with the release of free IAA from the
sugar esters tested, although a small activity was
observed using IAA-ester conjugates. This result may
indicate an important parallel or redundant role for
IBA conjugate hydrolysis in growth induction. One
strategy to regulate IAA-ester conjugates occurs in
maize, where it was demonstrated that the enzyme
catalyzing the conjugation of IAA to myoinositol was
also able to catalyze the hydrolysis of the IAA-ester
conjugate (Kowalczyk et al., 2003). Amide-type auxin
conjugates, including acyl peptides and proteins (Walz
et al. 2002), were not found in wheat seeds (Bandurski
and Schulze, 1977), but their occurrence in vegetative
tissues cannot be ruled out.
There appears to be a contradiction between the
temporal expression data of TaIAR3, suggesting the
transcript decreases in expression over 20 d, and the
enzymatic analysis data, suggesting an increase in
enzymatic activity as the seedlings age. This may
simply indicate a difference in how plants grown
under varying environmental conditions express the
enzyme, or this phenomenon could result from
the disparities in the growth conditions causing different physiological states between various types of
experiments. In addition, the TaIAR3 protein may be
Plant Physiol. Vol. 135, 2004
stable and accumulate in seedlings while the transcript
level is being down-regulated, or there may be posttranscriptional regulatory mechanisms involved.
Spatial expression of TaIAR3 indicates that, in seedlings and adult plants, the transcript is expressed at
approximately the same level in most tissues examined, supporting the belief that the enzyme is required
in some maintenance capacity in all tissues over the
life of the plant. However, loss-of-function mutants for
IAR3 in Arabidopsis are phenotypically wild type,
suggesting that IAR3 is not required for early development or mature growth (Davies et al., 1999).
Experiments are under way to isolate and characterize additional wheat hydrolase orthologs and also
orthologs from maize so that we may determine the
roles of each and their particular substrate specificity.
Functional characterization in future studies will provide essential information on how concentrations of
the different auxins may be regulated in our model
wheat and how this reflects on other plant species.
MATERIALS AND METHODS
Plant Materials and Plant Growth
Winter wheat (Triticum aestivum cv Caledonia) seeds were obtained
commercially from Johnny’s Selected Seeds (Albion, Maine). Plants were
raised under conditions optimized to fit the needs of the individual experiments performed, avoiding, in the interpretations, quantitative comparisons
between batches cultivated in different ways.
Seeds for sources of root, upper and lower leaf, and stem tissue were sown
in soil (perlite:sphagnum peat moss:vermiculite, 1:1:1, v/v/v) saturated with
liquid minimal medium (5 mM KNO3, 2.5 mM K2HPO4, pH 5.5, 2 mM MgSO4,
2 mM Ca(NO3)2.H2O, 50 mM Fe-EDTA, 70 mM H3BO3, 14 mM MnCl2, 0.5 mM
CuSO4, 1 mM ZnSO4, 0.2 mM Na2MoO4.2H2O, 10 mM NaCl, 0.01 mM CoCl2).
Plants were slowly hardened off over 1 week and fertilized with liquid
minimal medium every 2 to 3 weeks as needed. The plants were grown at 23°C
in constant light (cool-white, fluorescent, approximately 100 mmol s21 m22) in
a plant growth chamber (Model E-30B; Percival Scientific, Perry, IA).
Wheat seeds for harvest of coleoptile and radicle tissues were germinated
in 250-mL flasks with 50 mL of liquid Murashige and Skoog medium (SigmaAldrich, St. Louis). The flasks were agitated at approximately 100 rpm at 23°C
in constant light (cool-white, fluorescent, approximately 100 mmol s21 m22) in
a plant growth chamber (Model E-30B; Percival Scientific).
Leaves, roots, and stems for spatial expression analysis were harvested
70 d after germination. Coleoptiles and radicles were harvested at 4 d after
germination. All tissues were stored frozen at 280°C until RNA extraction.
For developmental expression studies in seedlings, seeds were germinated
in 250-mL flasks with 50 mL of liquid Murashige and Skoog medium. The
flasks were agitated at approximately 100 rpm at 23°C in constant light
in a plant growth chamber. Seedlings were collected 1, 2, 3, 4, 5, 10, 15, and
20 d after germination and stored frozen at 280°C until RNA extraction.
Wheat seeds used in enzyme activity measurement and endogenous auxin
determination were placed on moist filter paper, covered with plastic, and
incubated for 48 h at 4°C. The seeds were then placed at 23°C (60% humidity)
with a 16-h day/8-h night cycle. Day 1 after germination was specified as the
point when the radicles were first visible. The seedlings were then placed in
moist lecaton (expanded clay substrate) and harvested after the appropriate
time period after germination. Seedlings were watered every 2 d.
RNA Extraction
Total RNA was extracted from approximately 0.2 g of plant tissue, using
the RNeasy RNA extraction kit (Qiagen, Valencia, CA). Before extraction,
micropestles and all microfuge tubes were treated with an 8% solution of RNA
Secure (Ambion, Austin TX) for 10 min at 65°C. RNA concentration was
determined by UV absorbance (Spectronic Genesys 5 spectrophotometer;
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Campanella et al.
Thermo Electron, Waltham, MA) and samples stored at 280°C as separate
aliquots until real-time reverse transcription (RT)-PCR analysis could be
performed.
Cloning of Full-Length TaIAR3 cDNA
The cDNA sequence for TaIAR3 (T. aestivum TIGR accession no. TC150449,
GenBank accession no. AY701776) was obtained from TIGR sequence database
and first identified as an IAR3 ortholog (Sambrook et al., 1989). Total wheat
mRNA was extracted from whole seedlings and RT-PCR was then used to
amplify the specific cDNA of TaIAR3. The transcript primers used to amplify
TaIAR3 for cloning were 5#-AGAACGTCACGGCGGAATT-3# (TaIAR3F) and
5#-CAAACAGTTGCATAATCACCTG-3# (TaIAR3R). A single 50-mL reaction
was carried out in an RNase-free 0.5-mL microfuge tube using a Mastercycler
gradient thermocycler (Eppendorf Scientific,Westbury, NY). The RT reaction
was incubated at 50°C for 1 h, followed by 95°C for 15 min. The PCR step was
performed for 36 cycles at the following times and temperatures: 45 s at 95°C,
45 s at 57°C, and 1 min at 72°C.
The resulting amplified cDNA was blunt-end ligated, in-frame, into the
EcoRV cloning site of the pETBlue-2 expression vector (Novagen, Madison,
WI) using T4 DNA ligase (Novagen) in such a way that the cDNA was
terminated by its own stop codon and the His-tag of the pETBlue-2 vector was
not expressed. The resulting construct, peTaIAR3, was then transformed into
Escherichia coli (NovaBlue) using heat shock (Sambrook et al., 1989) and
putative transformants selected on the basis of ampicillin resistance and bluewhite selection (Sambrook et al., 1989). Plasmids were obtained from transformants by alkaline lysis (Sambrook et al., 1989). Insert orientation and size
were determined by endonuclease double digestion with BglI and BglII,
electrophoretic analyses through 1% agarose gels, and DNA sequencing of the
insert region.
The DNA/protein alignment and similarity calculations were performed
using the MatGAT v2.0 computer program (Campanella et al., 2003b). All
analyses were performed using default values.
Real-Time RT-PCR
Total RNA from wheat plants was used for real-time RT-PCR to examine
the expression of the TaIAR3 hydrolase gene. Analyses were performed on
two biological replicates for each treatment. The transcript-specific primers
used to amplify TaIAR3 were 5#-GTGGTGGAGCCTTCAATGTT-3# (TaIAR3
Exp F) and 5#-CAAACAGTTGCATAATCACCTG-3# (TaIAR3R). As an expression control for use in quantitation, universal 18S primers (Ambion) were
included in the same reaction mixes. This mixture of two sets of primers
constituted a duplexed RT-PCR reaction in which the primers were able to
amplify two different transcripts without interfering with each other (data not
shown).
All quantitative real-time analyses were performed as single-tube reactions
using approximately 1,500 ng of wheat mRNA with components of a One-Step
RT-PCR kit (Qiagen).This single 50-mL reaction was carried out in an RNasefree 0.5-mL microfuge tube using a Mastercycler gradient thermocycler
(Eppendorf). The RT reaction was incubated at 50°C for 1 h, followed by
95°C for 15 min. At the end of the RT reaction, 18S competimer primers
(Ambion) were added to the reaction tube to ensure that the abundant 18S
transcript did not overwhelm the hydrolase transcripts in amplification. The
ratio of 18S primers to 18S competimers in the reaction was 3:7.
The PCR step was performed for 32 to 44 cycles at the following times and
temperatures: 45 s at 95°C, 45 s at 57°C, and 1 min at 72°C. Ten 5-mL aliquots
were removed from the reaction tubes at the even-numbered cycles, starting at
cycle 14, 18, 20, 22, or 24, depending on the profile of initially detected
expression in each sample studied. After sampling, the reaction tube was
placed back on the thermocycler, during temperature ramping between
cycles, to proceed with PCR. Each 5-mL aliquot was frozen at 220°C and
stored for analysis.
The aliquots were analyzed by agarose gel electrophoresis and stained
with ethidium bromide. The RT-PCR products were imaged using an Ultralum gel documentation system (Ultralum, Claremont, CA) and Scion computer software (Scion Pharmaceuticals, Medford, MA). Densitometry was
performed on each cDNA band by application of the ImageTool Analysis
program (University of Texas Health Science Center, San Antonio). Each
experiment was repeated two to three times and densitometry values
averaged.
2236
The bar graphs indicating TaIAR3 expression were generated from the
real-time RT-PCR data. The standard curve method described by Applied
Biosystems (2001) was employed for this analysis. In this method, product
amounts were normalized to the 18S standard and a relative standard curve
was generated employing a basis sample, called a calibrator. For all experimental samples, target quantity was determined from the standard curve and
divided by the target quantity of the calibrator. The calibrator acted as the 13
sample, and all other samples were expressed in differences relative to the
calibrator.
Enzyme Preparation from Bacteria
The peTaIAR3 strain (containing the wheat homolog of IAR3 in the EcoRV
site of the pETBlue-2 vector) was grown overnight in 5 mL Luria-Bertani
medium containing 100 mg mL21 ampicillin. From this culture, 2 mL were
transferred to a flask containing 50 mL Luria-Bertani medium including
100 mg mL21 ampicillin and 1 mM IPTG for gene induction. Induction was
performed for 4 h under continuous shaking of the cultures. Uninduced
controls were grown under the same conditions, but without IPTG. Instead,
0.5% Glc was included in the medium since the promoter of pETBlue-2 is
leaky.
Enzyme preparation and enzyme assays with TaIAR3 were expressed in E.
coli (Campanella et al., 2003d). After collecting the bacterial cells by centrifugation for 10 min at 8,000g, the pellet was resuspended in 100 mL lysozyme
buffer per initial 1 mL bacterial culture (30 mM Tris-HCl, pH 8.0, containing
1 mM EDTA, 20% Suc, and 1 mg mL21 lysozyme [Sigma]) for cell lysis and
incubated for 10 min at 4°C. To break the cells, three freeze-thaw cycles were
performed where the cells were frozen in liquid N2 and thawed at 30°C. After
the last thawing, 2 mL of a 10 mg mL21 DNase solution (in 150 mM NaCl, 50%
glycerol) were added, and the mixture was incubated for 15 min at RT. The
extract (100-mL volume per assay) was then directly used for the enzyme
assay.
Enzyme Assay with TaIAR3
The enzyme assay for the hydrolysis of auxin conjugates was performed in
a 500-mL reaction mixture containing 395 mL assay buffer, 100 mL bacterial
enzyme extract (corresponding to approximately 2.5 mg total protein), and
5 mL of a 10 mM stock solution (dissolved in a small volume of ethanol, then
diluted with H2O) of each substrate (final concentration 100 mM; ethanol
concentration was always less then 0.1%). The substrates used in this study
were the IAA-amide conjugates IAA-Asp, IAA-Ala, IAA-Gly, IAA-Leu, IAAIle, IAA-Phe, and IAA-Val (all from Sigma), the IAA-ester conjugates IAA-Glc
and IAA-myoinositol (both gifts from Dr. Jerry D. Cohen), the amide conjugate
of IPA with Ala (synthesis described below), the amide conjugates of IBA with
Ala (Sigma) and Gly (obtained after demethylation of methyl-IBA-Gly, see
below), and the methyl esters of IBA-Ala and IBA-Gly (obtained from
Dr. Joseph Riov, The Volcani Center, Bet Dagan, Israel).
The assay buffer consisted of 100 mM Tris, pH 8.0, 10 mM MgCl2, 100 mM
MnCl2, 50 mM KCl, 100 mM PMSF, 1 mM DTT, and 10% Suc (Ludwig-Müller
et al., 1996). The reaction was routinely incubated for 1 h at 40°C. The reaction
was stopped by adding 100 mL 1 N HCl, and the aqueous phase was then
extracted with 600 mL ethyl acetate. The organic phase was removed,
evaporated to dryness, and resuspended in 100 mL CH3OH for HPLC analysis.
For GC-MS analysis, the samples were evaporated to dryness, redissolved in
100 mL ethyl acetate, methylated with diazomethane (Cohen, 1984), and
resuspended in ethyl acetate. The experiments were repeated three to five
times using different enzyme preparations. All results represent means of
independent experiments 6SE. As controls, the uninduced cultures were
evaluated. The enzymatic activity was calculated as nanomolar IAA released
from cultures induced with IPTG, subtracted by the values obtained in
noninduced cultures.
HPLC Analysis
The total CH3OH extract (100 mL, see above) was subjected to HPLC (BT
8100 pumps; Jasco, Easton, MD) coupled to an autosampler (AS-1550; Jasco),
equipped with a 125-mm 3 4.6-mm i.d. Lichrosorb C18 (particle size 5 mm)
reverse-phase column and a multiwavelength diode array detector (MC-919;
Jasco) set at 280 nm. As solvent, 1% aqueous CH3COOH (solvent A) and 100%
CH3OH (solvent B) were used. The program used was: 0 min, 25% B; 20 min,
25% B; 10 min, 100% B; 5 min, 25% B; 5 min equilibration, 25% B. Flow rate was
Plant Physiol. Vol. 135, 2004
Isolation of an Auxin Amidohydrolase from Wheat
1 mL min21. The BORWIN chromatography software (JMBS Developments
Software for Scientists; Varian, Palo Alto, CA) was used. Identification of IAA,
IBA, and IPA was achieved by comparison with authentic standards. IAA was
separated in this system from all conjugates used as substrates with a Rt of
23.4 min (Ludwig-Müller et al., 1996). The amount of the free IAA, IPA, or IBA
released was determined using a standard curve for the respective standard
substance.
GC-MS Analysis
GC-MS analysis was carried out on a Varian Saturn 2100 ion-trap mass
spectrometer using electron impact ionization at 70 eV, connected to a Varian
CP-3900 gas chromatograph equipped with a CP-8400 autosampler (Varian).
For the analysis, 2.5 mL of the methylated sample dissolved in 20 mL ethyl
acetate was injected in the splitless mode (splitter opening 1:100 after 1 min)
onto a Phenomenex ZB-5 column, 30 m 3 0.25 mm i.d. 3 0.25 mm using He
carrier gas at 1 mL min21. The injector temperature was 250°C and the
temperature program was 70° for 1 min, followed by an increase of 20° min21
to 280°C, then 5 min isothermically at 280°C. Transfer line temperature was
280°C. Either full-scan mass spectra were recorded or, for higher sensitivity,
the mSIS mode (Varian Manual) was used monitoring ions 130 and 189 (for
IAA methyl ester) or 217 (for IBA methyl ester). The scan rate was 0.6 s scan21,
the multiplier offset voltage was 200 V, the emission current was 30 mA, and
the trap temperature was 200°C. To identify the IBA enzymatically released
from its conjugates, the respective peak from HPLC was collected, evaporated,
methylated, and analyzed by GC-MS. IBA from the sample was identified
according to the Rt on GC compared with an authentic methylated standard
and by recording the respective mass spectrum.
Methylation and Demethylation of IBA Conjugates
To test whether the methyl esters of IBA amino acid conjugates were also
hydrolyzed, IBA-Ala was methylated using diazomethane. Briefly, the appropriate concentration of the conjugate was dissolved in 50 mL ethyl acetate, then
950 mL of a freshly prepared solution of diazomethane in diethyl ether were
added, and the mixture was incubated for 15 min at room temperature
(Cohen, 1984) The residual ether was evaporated and the methylated
compound dissolved in a small volume of CH3OH, or directly in ethyl acetate,
for GC-MS. The methanolic extract of Me-IBA-Ala was checked on HPLC in
an isocratic system (1% aqueous CH3COOH:CH3OH, 40:60, v/v), at a flow rate
of 0.7 mL min21 (Rt IBA-Ala, 8.8 min; Rt IBA-Ala methyl ester, 22.8 min). It
was shown that no unreacted IBA-Ala was present as an impurity (data not
shown).
The methyl ester of IBA-Gly was demethylated to yield IBA-Gly by
dissolving 100 mM methyl ester of IBA-Gly in 70% CH3OH. To this solution,
20 mL of a 2 N NaOH solution was added (final pH approximately 10). The pH
was monitored over the reaction time and was readjusted, if necessary. The
progress of the reaction was checked by thin-layer chromatography (TLC),
using silica gel plates (Merck, Rahway, NJ) and ethyl acetate:isopropanol:
NH4OHconc (45:35:20, v/v/v) as solvent. The methyl ester of IBA-Gly had an
RF value of approximately 1, IBA-Gly approximately 0.5. After 6-h reaction
time, the reaction did not proceed further and was therefore stopped by evaporating the CH3OH and adding 2 N HCl to the aqueous phase until the pH was
adjusted to 1.5. The H2O phase was extracted four times with equal volumes
of ethyl acetate. The organic phases were combined, dried over anhydrous
Na2SO4, evaporated to dryness, and resuspended in CH3OH. The IBA-Gly
was purified by HPLC. The HPLC system used for the purification of IBA-Gly
was an isocratic system (1% aqueous CH3COOH:CH3OH, 40:60, v/v), at a
flow rate of 0.7 mL min21 (Rt methyl ester of IBA-Gly, 21.4 min; Rt IBA-Gly,
7.3 min). The peak corresponding to IBA-Gly was collected, evaporated to
dryness, and resuspended in a small volume of CH3OH. For enzymatic assays,
this methanolic extract was diluted with H2O as for the other auxin
conjugates.
Synthesis of N-[3-(Indol-3-yl)propionyl]-L-Ala
In the syntheses, commercial, analytical grade chemicals and solvents were
used without further purification. Only dioxane was redistilled over sodium,
immediately before use, to ensure the absence of peroxides. Melting points
were determined in open capillaries and were not corrected. High-resolution
mass spectra were obtained on a Micromass Q-TOF2 instrument using
electrospray ionization (positive ion mode; capillary voltage, 3 kV; cone
Plant Physiol. Vol. 135, 2004
voltage, 30 V; source temperature, 80°C; desolvation temperature, 150°C;
nitrogen flow, 480 L h21; sample applied in acetonitrile:water, 2:8, containing
0.1% formic acid). Nuclear magnetic resonance (NMR) spectra were taken on
a Bruker AV600 instrument operating at 600 MHz for 1H and at 150 MHz for
13
C. Chemical shifts are reported in parts per million relative to internal
tetramethylsilane. TLC was performed on glass plates (5 3 10 cm), coated
with silica gel GF254 (Merck), to a thickness of 0.3 mm. Chromatograms were
developed with solvents A (2-propanol:ethyl acetate:NH4OHconc, 35:45:20,
v/v/v) and B (CH2Cl2:CH3OH:CH3COOH, 90:10:5, v/v/v). Detection was by
UV absorbance and by spraying with Ehrlich’s reagent (1% [w/v] 4-dimethylaminobenzaldehyde in a 1:1 [v/v] mixture of ethanol and concentrated HCl),
which affords blue to purple spots with the indole derivatives studied here.
To a stirred solution of 3-(indol-3-yl) propionic acid (1 g, 5.29 mmol) and
N-hydroxysuccinimide (639 mg, 5.55 mmol) in a mixture of dioxane (6 mL)
and anhydrous ethyl acetate (4 mL), 1,3-dicyclohexylcarbodiimide (1.20 g,
5.81 mmol) was added in small aliquots, for 20 min, at 28°C. After 2 h of
further stirring in an ice-water bath, TLC (solvents A and B) indicated that the
reaction was complete. The precipitated 1,3-dicyclohexylurea was removed by
filtration and rinsed with ethyl acetate. The filtrate was evaporated to yield the
crude 3-(indol-3-yl) propionic acid N-hydroxysuccinimide ester, RF 5 0.6
(solvent A) and 0.4 (solvent B). The latter was redissolved in dioxane (20 mL),
a solution of L-Ala (471 mg, 5.29 mmol) in 10% aqueous NaHCO3 (20 mL) was
added, and the resulting suspension was stirred at room temperature, for 1 h,
when TLC (solvent A) indicated that the reaction was complete. The mixture
was diluted with water (20 mL, pH after dilution, 9.6) and nonacidic byproducts were extracted with diethyl ether (2 3 30 mL). The aqueous phase
was acidified to pH 2.5 and partitioned against ethyl acetate (5 3 50 mL). The
organic phase was dried over anhydrous sodium sulfate and evaporated to
yield the crude title compound. Repeated crystallization from ethyl acetate:
cyclohexane (approximately 1:1, v/v) afforded off-white crystals (1.02 g, 74%),
RF 5 0.4 (solvent A), melting point 179°C to 181°C. High-resolution mass
spectrum (time-of-flight instrument, electron spray ionization with positive
ion monitoring): 261.1218 (MH1; C14H17N2O3 requires 261.1239), 172.0701 ([M
- Ala]1; C11H10NO requires 172.0762), 130.0655 (quinolinium ion; C9H8N
requires 130.0657) amu. 1H-NMR spectrum [(CD3)2SO]: 3-(indol-3-yl) propionyl moiety: d 10.71 (1 H, s, H-1), 7.06 (1 H, narrow m, H-2), 7.47 (1 H, d, J4,5
5 7.8 Hz, H-4), 6.91 (1 H, t, J5,6 5 7.1 Hz, H-5), 7.00 (1 H, t, H-6), 7.27 (1 H, d, J6,7
5 8.1 Hz, H-7), 2.42 (1 H, dt, Jgem 5 14.7 Hz, Jvic 5 7.6 Hz, ArCHI), 2.46 (1 H, dt,
ArCHII), 2.87 (2 H, t, CH2CO); L-Ala moiety: d 8.16 (1 H, d, JCH,NH 5 7.2 Hz,
NH), 4.19 (1 H, quintet, JCH2,CH3 5 7.3 Hz, CH), 1.20 (1 H, t, CH3), 12.46
(1 H, broad s, COOH). 13C-NMR spectrum [(CD3)2SO]: 3-(indol-3-yl)
propionyl moiety: d 122.2 (C-2), 113.9 (C-3), 127.1 (C-3a), 118.3 (C-4), 118.2
(C-5), 120.9 (C-6), 111.3 (C-7), 136.3 (C-7a), 20.9 (ArCH2), 36.0 (CH2CO), 171.8
(CONH); L-Ala moiety d: 17.3 (CH3), 47.5 (CHNH), 174.3 (COOH).
Determination of Endogenous Auxins
Free and Conjugated IAA and IBA
Wheat seedlings were harvested after different times of culture (3, 6, 10, 15,
and 17 d after germination), the roots were washed thoroughly and the tissue
was blotted dry between sheets of filter paper. The plant material (approximately 0.1 g fresh weight per analysis) was extracted as previously described
(Chen et al., 1988; Ludwig-Müller and Cohen, 2002). Each extract was divided
into two equal parts for determination of free auxins and for alkaline
hydrolysis. To each sample, 200 ng 13C6-IAA (Cambridge Isotope Laboratories,
Andover, MS), and 100 ng 13C1-IBA (Sutter and Cohen, 1992) were added. For
each sample, three independent extractions were performed. Free auxins were
purified on NH2 columns (Chen et al., 1988) and, after elution from the
column, the samples were evaporated to dryness, directly methylated with
diazomethane (Cohen, 1984), and resuspended in ethyl acetate for GC-MS
analysis.
Hydrolysis of conjugated auxins was performed with 7 N NaOH at 100°C
under N2 for 3 h. The hydrolysate was filtered, the pH adjusted to 2.5, and the
auxins were extracted twice with equal volumes of ethyl acetate. The organic
phase was evaporated and the extract methylated as described above. GC-MS
analysis was performed with the program described above for the analysis of
enzymatically released IBA. Endogenous auxin concentrations were calculated using the isotope dilution equation (Ilić et al., 1996).
The experiments were performed three times using different plant extracts.
All results present the arithmetic means of independent experiments 6SE.
Statistical analysis of the data was done using analysis of variance (ANOVA)
2237
Campanella et al.
employing a Web-based program (http://www.physics.csbsju.edu/stats/
anova.html).
Identification of Endogenous IBA-Ala
The nonhydrolyzed methanolic extracts from wheat seedlings were subjected to GC-MS analysis after methylation with diazomethane (see above). As
a standard, the methyl ester of IBA-Ala was subjected to the same GC-MS
procedure. The settings used for separation were as described above, with the
following modifications: Injector temperature was 250°C and the temperature
program was 70°C for 2 min, followed by an increase of 10°C min21 to 300°C,
then 10 min isothermically at 300°C. Transfer line temperature was 300°C.
Endogenous IBA-Ala was identified according to the Rt of its methyl ester in
the ion chromatogram (26.1 min) and the respective mass spectrum was
recorded and compared to that of an authentic IBA-Ala standard.
In Vitro Assay for Auxin Conjugate Hydrolysis Using
Wheat Seedlings
The determination of enzymatic activity in plant extracts was carried out as
in Ludwig-Müller et al. (1996). The same buffer as for the enzymatic assays
with bacteria was used. Briefly, the seedlings were harvested for enzyme
preparation, the roots were thoroughly washed and then blotted dry between
sheets of filter paper. The plants were extracted in a 100 mM Tris-HCl buffer,
pH 8, containing 10 mM MgCl2, 100 mM MnCl2, 50 mM KCl, 1 mM DTT, 100 mM
PMSF, and 10% Suc. The homogenate was filtered and then centrifuged for
30 min at 50,000g. The supernatant was used for the hydrolase assays.
The enzyme assay for the conversion of IAA conjugates to free IAA was
performed in a 500-mL reaction mixture containing 300 mL of enzyme extract
and 50 mM of individual substrate (IAA-Ala and IBA-Ala). The enzyme
reaction was carried out at pH 8.0 for 60 min at 40°C, as described above.
Inactivated extracts (boiling for 10 min) were also incubated and used as
controls to determine nonenzymatic hydrolysis of auxin conjugates. These
values were subtracted from the values determined with active enzyme
extract. The reaction was stopped by adding 100 mL 1 N HCl, and the aqueous
phase was then extracted with 500 mL ethyl acetate. The organic phase was
removed, evaporated to dryness, and resuspended in 20 mL CH3OH. The
sample was kept in liquid nitrogen prior to HPLC analysis.
Protein was determined with the Bradford protein assay reagent (Sigma)
using bovine serum albumin as standard (Bradford, 1976).
The experiments were performed three times using different enzyme
preparations. All results represent the arithmetic means of independent
experiments 6SE.
Phylogenetic Tree Construction
Amino acid alignments of TaIAR3, ILR1, IAR3, ILL1, ILL2, ILL3, and ILL6
orthologs were performed using ClustalX v1.8 software (Thompson et al.,
1997). Alignment settings were employed at default values. Phylogenetic trees
were generated from the distances provided by the ClustalX analysis using
the neighbor-joining method (Saitou and Nei, 1987). Bootstrap analyses
(Felsenstein, 1985) consisted of 1,000 replicates. The neighbor-joining trees
were visualized with the TREEVIEW program (Page, 1996). The protein
sequence of a bacterial M20 peptidase from Campylobacter jejuni (GenBank
accession no. Z36940) was used as an outgroup.
Sequence data from this article have been deposited with the EMBL/
GenBank data libraries under accession number AY701776.
ACKNOWLEDGMENTS
We thank Dr. Joseph Riov, The Volcani Center, Bet Dagan, Israel, for the
gift of the methyl ester of IBA-Gly, Dr. Ellen G. Sutter, University of
California, Davis, for 13C1-IBA, Dr. Jerry D. Cohen, University of Minnesota,
St. Paul, for IAA-ester conjugates with Glc and myoinositol, and Igor Bratos,
PLIVA Pharmaceuticals, Zagreb, Croatia, for high-resolution mass spectra.
The helpful discussions with Dr. Ephraim Epstein are gratefully acknowledged. We thank Silvia Heinze and Dr. Campanella’s graduate molecular
biology laboratory class for technical assistance. Finally, we wish to thank
Lisa Campanella for her generous editorial help.
2238
Received March 23, 2004; returned for revision June 4, 2004; accepted June 7,
2004.
LITERATURE CITED
Albert VA, Oppenheimer DG, Lindqvist C (2002) Pleiotropy, redundancy
and the evolution of flowers. Trends Plant Sci 7: 297–301
Andersson B, Sandberg G (1982) Identification of endogenous N-(3indoleacetyl)aspartic acid in Scots pine (Pinus sylvestris L.) by combined
gas chromatography-mass spectrometry, using high-performance liquid
chromatography for quantification. J Chromatogr 238: 151–156
Applied Biosystems Incorporated (2001) User Bulletin #2: ABI PRISM 7700
Sequence Detection System. http://www.appliedbiosystems.com
Aung LH (1972) The nature of root-promoting substances in Lycopersicon
esculentum seedlings. Physiol Plant 26: 306–309
Bandurski RS, Cohen JD, Slovin JP, Reinecke DM (1995) Auxin biosynthesis and metabolism. In PJ Davies, ed, Plant Hormones: Physiology, Biochemistry and Molecular Biology, Ed 2. Kluwer Academic
Publishers, Dordrecht, The Netherlands, pp 39–65
Bandurski RS, Schulze A (1977) Concentrations of IAA and its derivatives
in plants. Plant Physiol 60: 211–213
Bandurski RS, Ueda M, Nicholls PB (1969) Esters of indole-3-acetic acid
and myo-inositol. Ann N Y Acad Sci 165: 655–667
Baraldi R, Bertazza G, Predieri S, Bregoli AM, Cohen JD (1993) Uptake
and metabolism of indole-3-butyric acid during the in vitro rooting
phase in pear cultivars (Pyrus communis). Acta Hortic 329: 289–291
Bartel B, Fink G (1995) ILR1, an amidohydrolase that releases active
indole-3-acetic acid from conjugates. Science 268: 1745–1748
Bayer M (1969) Gas chromatographic analysis of acidic indole auxins in
Nicotiana. Plant Physiol 44: 267–271
Bradford MM (1976) A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem 72: 248–254
Campanella JJ, Bakllamaja V, Restieri T, Vomacka M, Herron J, Patterson
M, Shahtaheri S (2003a) Isolation of an ILR1 auxin conjugate hydrolase
homolog from Arabidopsis suecica. Plant Growth Regul 39: 175–181
Campanella JJ, Bitincka L, Smalley J (2003b) MatGAT: an application that
generates similarity/identity matrices using protein or DNA sequences.
BMC Bioinformatics 4: 29
Campanella JJ, Larko D, Smalley J (2003c) A molecular phylogenomic
analysis of the ILR1-like family of IAA amidohydrolase genes. Comp
Funct Genomics 4: 584–600
Campanella JJ, Ludwig-Müller J, Bakllamaja V, Sharma V, Cartier A
(2003d) ILR1 and sILR1 IAA amidohydrolase homologs differ in
expression pattern and substrate specificity. Plant Growth Regul 41:
215–223
Campanella JJ, Ludwig-Müller J, Town CD (1996) Isolation and characterization of mutants of Arabidopsis thaliana with increased resistance to
growth inhibition by indoleacetic acid-amino acid conjugates. Plant
Physiol 112: 735–745
Chen KH, Miller AN, Patterson GW, Cohen JD (1988) A rapid and simple
procedure for purification of indole-3-acetic acid prior to GC-SIM-MS
analysis. Plant Physiol 86: 822–825
Chhun T, Taketa S, Tsurumi S, Ichii M (2003) The effects of auxin on lateral
root initiation and root gravitropism in a lateral rootless mutant Lrt1 of
rice (Oryza sativa L.). Plant Growth Regul 39: 161–170
Chisnell JR, Bandurski RS (1988) Translocation of radiolabeled indole-3acetic acid and indole-3-acetyl-myo-inositol from kernel to shoot in Zea
mays L. Plant Physiol 86: 79–84
Cohen JD (1982) Identification and quantitative analysis of indole-3-acetylL-aspartate from seeds of Glycine max L. Plant Physiol 70: 749–753
Cohen JD (1984) Convenient apparatus for the generation of small amounts
of diazomethane. J Chromatogr 303: 193–196
Cohen JD, Bandurski RS (1982) The chemistry and physiology of the
bound auxins. Annu Rev Plant Physiol 33: 403–430
Cooke TJ, Poli DB, Cohen JD (2004) Did auxin play a crucial role in the
evolution of novel body plans during the Late-Silurian-Early Devonian
radiation of land plants? In AR Hemsley, I Poole, eds, The Evolution of
Plant Physiology: From Whole Plants to Ecosystems. Linnean Society of
London and Elsevier Academic Press, Amsterdam, pp 85–107
Cooke TJ, Poli DB, Sztein AE, Cohen JD (2002) Evolutionary patterns in
auxin action. Plant Mol Biol 49: 319–338
Plant Physiol. Vol. 135, 2004
Isolation of an Auxin Amidohydrolase from Wheat
Davies PJ (1995) The plant hormones: their nature, occurrence, and
functions, In PJ Davies, ed, Plant Hormones: Physiology, Biochemistry,
and Molecular Biology. Kluwer Academic Publishers, Dordrecht, The
Netherlands, pp 13–38
Davies RT, Goetz DH, Lasswell J, Anderson MN, Bartel B (1999) IAR3
encodes an auxin conjugate hydrolase from Arabidopsis. Plant Cell 11:
365–376
Domagalski W, Schulze A, Bandurski RS (1987) Isolation and characterization of esters of indole-3-acetic acid from liquid endosperm of the
horse chestnut (Aesculus species). Plant Physiol 84: 1107–1113
Elsden SR, Hilton MG, Waller JM (1976) The end products of the
metabolism of aromatic amino acids by Clostridia. Arch Microbiol
107: 283–288
Epstein E, Chen K-H, Cohen JD (1989) Identification of indole-3-butyric
acid as an endogenous constituent of maize kernels and leaves. Plant
Growth Regul 8: 215–223
Epstein E, Lavee S (1984) Conversion of indole-3-butyric acid to indole-3acetic acid by cuttings of grapevine (Vitis vinifera) and olive (Olea
europea). Plant Cell Physiol 25: 692–703
Epstein E, Ludwig-Müller J (1993) Indole-3-butyric acid in plants: occurrence, synthesis, metabolism and transport. Physiol Plant 88: 382–389
Fawcett CH, Wain RI, Wightman F (1960) The metabolism of 3-indolylalkanecarboxylic acids, and their amides, nitriles and methyl esters in
plant tissues. Proc R Soc Lond B Biol Sci 152: 231–254
Felsenstein J (1985) Confidence limits on phylogenies: an approach using
the bootstrap. Evolution 39: 783–791
Hall PJ, Bandurski RS (1986) [3H]Indole-3-acetyl-myo-inositol hydrolysis
by extracts of Zea mays L. vegetative tissue. Plant Physiol 80: 374–377
Hartmann HT, Kester DE, Davies FT (1990) Plant Propagation: Principles
and Practices. Prentice-Hall, Englewood Cliffs, NJ, pp 246–247
Ilić N, Normanly J, Cohen JD (1996) Quantification of free plus conjugated
indole-3-acetic acid in Arabidopsis requires correction for the nonenzymatic conversion of indolic nitriles. Plant Physiol 111: 781–788
Katekar G (1979) Auxins: on the nature of the receptor site and molecular
requirements for auxin activity. Phytochemistry 18: 223–233
Kellogg EA (2001) Evolutionary history of the grasses. Plant Physiol 125:
1198–1205
Kopcewicz J, Ehmann A, Bandurski RS (1974) Enzymatic esterification
of indole-3-acetic acid to myo-inositol and glucose. Plant Physiol 54:
846–851
Kowalczyk M, Sandberg G (2001) Quantitative analysis of indole-3-acetic
acid metabolites of Arabidopsis. Plant Physiol 127: 1845–1853
Kowalczyk S, Jakubowska A, Zielinska E, Bandurski RS (2003) Bifunctional indole-3-acetyl transferase catalyses synthesis and hydrolysis of
indole-3-acetyl-myo-inositol in immature endosperm of Zea mays. Physiol Plant 119: 165–174
LeClere S, Tellez R, Rampey RA, Matsuda SPT, Bartel B (2002) Characterization of a family of IAA-amino acid conjugate hydrolases from
Arabidopsis. J Biol Chem 277: 20446–20452
Linser H, Mayr H, Maschek F (1954) Papierchromatographie von
zellstreckend wirksamen Indolkörpern aus Brassica-.Arten. Planta 44:
103–120
Ludwig-Müller J (2000) Indole-3-butyric acid in plant growth and development. Plant Growth Regul 32: 219–230
Ludwig-Müller J, Cohen JD (2002) Identification and quantification of
three active auxins in different tissues of Tropaeolum majus. Physiol Plant
115: 320–329
Ludwig-Müller J, Epstein E (1991) Occurrence and in vivo biosynthesis of
indole-3-butyric acid in corn (Zea mays L.). Plant Physiol 97: 765–770
Ludwig-Müller J, Epstein E (1993) Indole-3-butyric acid in Arabidopsis
thaliana. II. In vivo metabolism. Plant Growth Regul 13: 189–195
Ludwig-Müller J, Epstein E, Hilgenberg W (1996) Auxin-conjugate
hydrolysis in Chinese cabbage: characterization of an amidohydrolase
and its role during infection with clubroot disease. Physiol Plant 97:
627–634
Ludwig-Müller J, Hilgenberg W (1995) Characterization and partial
purification of indole-3-butyric acid synthetase from maize (Zea mays).
Physiol Plant 94: 651–660
Ludwig-Müller J, Hilgenberg W, Epstein E (1995a) The in vitro biosynthesis of indole-3-butyric acid in maize. Phytochemistry 40: 61–68
Ludwig-Müller J, Raisig A, Hilgenberg W (1995b) Uptake and transport of
indole-3-butyric acid in Arabidopsis thaliana: comparison with other
natural and synthetic auxins. J Plant Physiol 147: 351–354
Plant Physiol. Vol. 135, 2004
Ludwig-Müller J, Sass S, Sutter EG, Wodner M, Epstein E (1993) Indole-3butyric acid in Arabidopsis thaliana. I. Identification and quantification.
Plant Growth Regul 13: 179–187
Ludwig-Müller J, Schubert B, Pieper K (1995c) Regulation of IBA
synthetase from maize (Zea mays L.) by drought stress and ABA. J Exp
Bot 46: 423–432
Mapelli S, Locatelli F, Bertani A (1995) Effect of anaerobic environment on
germination and growth of rice and wheat: endogenous levels of ABA
and IAA. Bulg J Plant Physiol 21: 33–41
Matsuda K, Toyoda H, Nishio H, Nishida T, Dohgo M, Bingo M, Matsuda
Y, Yoshida S, Harada S, Tanaka H, et al (1998) Control of the bacterial
wilt of tomato plants by a derivative of 3-indolepropionic acid based on
selective actions on Ralstonia solanacearum. J Agric Food Chem 46: 4416–
4419
Mohammed N, Onodera R, Or-Rashid MM (2003) Degradation of tryptophan and related indolic compounds by ruminal bacteria, protozoa and
their mixture in vitro. Amino Acids 24: 73–80
Nordström AC, Jacobs FA, Eliasson L (1991) Effect of exogenous indole-3acetic acid and indole-3-butyric acid on internal levels of the respective
auxins and their conjugation with aspartic acid during adventitious root
formation in pea cuttings. Plant Physiol 96: 856–861
Oliviusson P, Salaj J, Hakman I (2001) Expression pattern of transcripts
encoding water channel-like proteins in Norway spruce. Plant Mol Biol
46: 289–299
Östin A, Moritz T, Sandberg G (1992) Liquid chromatography/mass
spectrometry of conjugates and oxidative metabolites of indole-3-acetic
acid. Biol Mass Spectrom 21: 292–298
Page RD (1996) TREEVIEW: an application to display phylogenetic trees on
personal computers. Comput Appl Biosci 12: 357–358
Park S, Walz A, Momonoki YS, Ludwig-Müller J, Slovin JP, Cohen JD
(2003) Partial characterization of major IAA conjugates in Arabidopsis.
Proceedings of the American Society of Plant Biologists Conference, July
25–30, 2003, Honolulu, HI. http://abstracts.aspb.org/pb2003/public/
P46/0961.html
Rampey RA, LeClere S, Bartel B (2003) The roles of Arabidopsis IAA
conjugate hydrolases in auxin homeostasis. Proceedings of the American Society of Plant Biologists Conference, July 25–30, 2003, Honolulu,
HI. http://abstracts.aspb.org/pb2003/public/P46/0446.html
Saitou N, Nei M (1987) The neighbor-joining method: a new method for
reconstructing phylogenetic trees. Mol Biol Evol 4: 406–425
Sambrook J, Maniatis T, Fritsch EF (1989) Molecular Cloning: A Laboratory
Manual, Ed 2. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Schneider EA, Kazakoff CW, Wightman F (1985) Gas chromatographymass spectrometry evidence for several endogenous auxins in pea
seedling organs. Planta 165: 232–241
Schuller A, Ludwig-Müller J (2002) Isolation of differentially expressed
genes involved in clubroot disease. Plant Prot Sci 38: 483–486
Segal LM, Wightman F (1982) Gas chromatographic and GC-MS evidence
for the occurrence of 3-indolylpropionic acid and 3-indolylacetic acid in
seedlings of Cucurbita pepo. Physiol Plant 56: 367–370
Slovin JP, Bandurski RS, Cohen JD (1999) Auxins. In PJJ Hooykaas, MA
Hall, KR Libbenga, eds, Biochemistry and Molecular Biology of Plant
Hormones. Elsevier, Amsterdam, The Netherlands, pp 115–140
Sonner JM, Purves WK (1985) Natural occurrence of indole-3-acetyl
aspartate and indole-3-acetyl glutamate in cucumber shoot tissue. Plant
Physiol 77: 784–785
Sutter EG, Cohen JD (1992) Measurement of indolebutyric acid in plant
tissues by isotope dilution gas chromatography-mass spectrometry
analysis. Plant Physiol 99: 1719–1722
Tabone D (1958) Biosynthèse par B. megatherium de combinaisons de
l’acide indol propionique avec certains acides aminés. Bull Soc Chim
Biol (Paris) 40: 965–969
Tabone J, Tabone D (1953) Bio-estérification du glucose. V. Bio-synthèse
par Bacillus megatherium de l’ester b glucosidique de l’acide indolpropionique. C R Hebd Seances Acad Sci 237: 943–944
Tam YY, Epstein E, Normanly J (2000) Characterization of auxin conjugates
in Arabidopsis. Low steady-state levels of indole-3-acetyl aspartate,
indole-3-acetyl glutamate, and indole-3-acetyl glucose. Plant Physiol
123: 589–595
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997)
The ClustalX Windows interface – flexible strategies for multiple
sequence alignment aided by quality analysis tools. Nucleic Acids Res
25: 4876–4882
2239
Campanella et al.
Troitsky AV, Melekhovets YF, Rakhimova GM, Bobrova VK, ValiejoRoman KM, Antonov AS (1991) Angiosperm origin and early stages of
seed plant evolution deduced from rRNA sequence comparisons. J Mol
Evol 32: 253–261
Van der Krieken W, Bretler H, Visser M (1992) The effect of the conversion
of indolebutyric acid into indoleacetic acid on root formation on microcuttings of Malus. Plant Cell Physiol 33: 709–713
Walker TS, Bais HP, Halligan KM, Stermitz FR, Vivanco JM (2003)
Metabolic profiling of root exudates of Arabidopsis thaliana. J Agric Food
Chem 51: 2548–2554
Walz A, Park S, Slovin JP, Ludwig-Müller J, Momonoki YS, Cohen JD
(2002) A gene encoding a protein modified by the phytohormone
indoleacetic acid. Proc Natl Acad Sci USA 99: 1718–1723
Wightman F, Schneider EA, Thimann KV (1980) Hormonal factors
controlling the initiation and development of lateral roots. II. Effects
2240
of exogenous growth factors on lateral root formation in pea roots.
Physiol Plant 49: 304–314
Wolfe KH, Gouy M, Yang YW, Sharpe PM, Li WH (1989) Date of the
monocot-dicot divergence estimated from chloroplast DNA sequence
data. Proc Natl Acad Sci USA 86: 6201–6205
Zolman BK, Bartel B (2004) An Arabidopsis indole-3-butyric-acid-response
mutant defective in PEROXIN6, an apparent ATPase implicated in
peroxisomal function. Proc Natl Acad Sci USA 101: 1786–1791
Zolman BK, Silva ID, Bartel B (2001) The Arabidopsis pxa1 mutant
is defective in an ATP-binding cassette transporter-like protein
required for peroxisomal fatty acid b-oxidation. Plant Physiol 127:
1266–1278
Zolman BK, Yoder A, Bartel B (2000) Genetic analysis of indole-3-butyric
acid responses in Arabidopsis thaliana reveals four mutant classes.
Genetics 156: 1323–1337
Plant Physiol. Vol. 135, 2004