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DEVELOPMENT AND HORMONE ACTION

A Novel Auxin Conjugate Hydrolase from Wheat with Substrate Specificity for Longer Side-Chain Auxin Amide Conjugates¹

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.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
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 IAA-myoinositol 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.

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 (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 IAA-myoinositol glycosides, IAA-myoinositol, and IAA-Glc (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 IAA-Gly 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, 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 IAA-inositol (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

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

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).

View larger version (9K): [in this window] [in a new window]   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.

View larger version (15K): [in this window] [in a new window]   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.

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-d-old 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 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.

View larger version (23K): [in this window] [in a new window]   Figure 3. Free and total IAA and IBA concentrations in wheat during seedling development. The values presented are means ± SE of three independent experiments.

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 IPA-Ala is not a native substance in wheat seedlings.

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).

View larger version (17K): [in this window] [in a new window]   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.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
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 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 Ludwig-Müller, 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 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 -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 (Ludwig-Müller and Epstein, 1991; Ludwig-Müller et al., 1995c).

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. Ludwig-Müller, 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 gymnosperm-angiosperm 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 IAA-ester 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 stable and accumulate in seedlings while the transcript level is being down-regulated, or there may be post-transcriptional 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

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

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 µM Fe-EDTA, 70 µM H₃BO₃, 14 µM MnCl₂, 0.5 µM CuSO₄, 1 µM ZnSO₄, 0.2 µM Na₂MoO₄.2H₂O, 10 µM NaCl, 0.01 µM 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 µmol s^–1 m^–2) 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 (Sigma-Aldrich, St. Louis). The flasks were agitated at approximately 100 rpm at 23°C in constant light (cool-white, fluorescent, approximately 100 µmol s^–1 m^–2) 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 –80°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 –80°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; Thermo Electron, Waltham, MA) and samples stored at –80°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-µL 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 blue-white 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-µL reaction was carried out in an RNase-free 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-µL 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-µL aliquot was frozen at –20°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.

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 1x 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 µg mL^–1 ampicillin. From this culture, 2 mL were transferred to a flask containing 50 mL Luria-Bertani medium including 100 µg mL^–1 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 µL lysozyme buffer per initial 1 mL bacterial culture (30 mM Tris-HCl, pH 8.0, containing 1 mM EDTA, 20% Suc, and 1 mg mL^–1 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 µL of a 10 mg mL^–1 DNase solution (in 150 mM NaCl, 50% glycerol) were added, and the mixture was incubated for 15 min at RT. The extract (100-µL 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-µL reaction mixture containing 395 µL assay buffer, 100 µL bacterial enzyme extract (corresponding to approximately 2.5 mg total protein), and 5 µL of a 10 mM stock solution (dissolved in a small volume of ethanol, then diluted with H₂O) of each substrate (final concentration 100 µM; 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, IAA-Ile, 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 µM MnCl₂, 50 mM KCl, 100 µM 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 µL 1 N HCl, and the aqueous phase was then extracted with 600 µL ethyl acetate. The organic phase was removed, evaporated to dryness, and resuspended in 100 µL CH₃OH for HPLC analysis. For GC-MS analysis, the samples were evaporated to dryness, redissolved in 100 µL 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 ±SE. 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 µL, see above) was subjected to HPLC (BT 8100 pumps; Jasco, Easton, MD) coupled to an autosampler (AS-1550; Jasco), equipped with a 125-mm x 4.6-mm i.d. Lichrosorb C₁₈ (particle size 5 µm) 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 1 mL min^–1. 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 µL of the methylated sample dissolved in 20 µL ethyl acetate was injected in the splitless mode (splitter opening 1:100 after 1 min) onto a Phenomenex ZB-5 column, 30 m x 0.25 mm i.d. x 0.25 µm using He carrier gas at 1 mL min^–1. The injector temperature was 250°C and the temperature program was 70° for 1 min, followed by an increase of 20° min^–1 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 µSIS 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 scan^–1, the multiplier offset voltage was 200 V, the emission current was 30 µA, 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 µL ethyl acetate, then 950 µL 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 min^–1 (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 µL 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:NH₄OH_conc (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 min^–1 (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 voltage, 30 V; source temperature, 80°C; desolvation temperature, 150°C; nitrogen flow, 480 L h^–1; 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 ¹³C. Chemical shifts are reported in parts per million relative to internal tetramethylsilane. TLC was performed on glass plates (5 x 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:NH₄OH_conc, 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 –8°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 = 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 by-products were extracted with diethyl ether (2 x 30 mL). The aqueous phase was acidified to pH 2.5 and partitioned against ethyl acetate (5 x 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 = 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 (MH⁺; C₁₄H₁₇N₂O₃ requires 261.1239), 172.0701 ([M - Ala]⁺; C₁₁H₁₀NO requires 172.0762), 130.0655 (quinolinium ion; C₉H₈N requires 130.0657) amu. ¹H-NMR spectrum [(CD₃)₂SO]: 3-(indol-3-yl) propionyl moiety: 10.71 (1 H, s, H-1), 7.06 (1 H, narrow m, H-2), 7.47 (1 H, d, J_4,5 = 7.8 Hz, H-4), 6.91 (1 H, t, J_5,6 = 7.1 Hz, H-5), 7.00 (1 H, t, H-6), 7.27 (1 H, d, J_6,7 = 8.1 Hz, H-7), 2.42 (1 H, dt, J_gem = 14.7 Hz, J_vic = 7.6 Hz, ArCH_I), 2.46 (1 H, dt, ArCH_II), 2.87 (2 H, t, CH₂CO); L-Ala moiety: 8.16 (1 H, d, J_CH,NH = 7.2 Hz, NH), 4.19 (1 H, quintet, J_CH2,CH3 = 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: 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 : 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 (Ili et al., 1996).

The experiments were performed three times using different plant extracts. All results present the arithmetic means of independent experiments ±SE. Statistical analysis of the data was done using analysis of variance (ANOVA) 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 min^–1 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 µM MnCl₂, 50 mM KCl, 1 mM DTT, 100 µM 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-µL reaction mixture containing 300 µL of enzyme extract and 50 µM 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 µL 1 N HCl, and the aqueous phase was then extracted with 500 µL ethyl acetate. The organic phase was removed, evaporated to dryness, and resuspended in 20 µL 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 ±SE.

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.

Received March 23, 2004; returned for revision June 4, 2004; accepted June 7, 2004.

	FOOTNOTES

 
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).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.043398.

^* Corresponding author; e-mail james.campanella{at}montclair.edu; fax 419–791–9834.

	LITERATURE CITED

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