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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.
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Plant Physiology, August 2004, Vol. 135, pp. 22302240, www.plantphysiol.org 2004 American Society of Plant Biologists
RESULTS
Substrate Specificity of TaIAR3
Campanella et al.
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.
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.
Age of Seedlings
IBA-Ala
days after germination
0
3
6
15
17
IAA-Ala
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.
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. LudwigMuller, 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
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 65C. RNA concentration was
determined by UV absorbance (Spectronic Genesys 5 spectrophotometer;
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Campanella et al.
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 50C for 1 h, followed by
95C 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 95C, 45 s at 57C, and 1 min at 72C. 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 220C 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.
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
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 250C and the
temperature program was 70 for 1 min, followed by an increase of 20 min21
to 280C, then 5 min isothermically at 280C. Transfer line temperature was
280C. 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 200C. 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.
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
2237
Campanella et al.
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. Campanellas 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.
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