Plant Growth Regulation 41: 215–223, 2003. # 2003 Kluwer Academic Publishers. Printed in the Netherlands.
215
ILR1 and sILR1 IAA amidohydrolase homologs differ in expression pattern and substrate specificity James J. Campanella1,*, Jutta Ludwig-Mueller2, Vinela Bakllamaja1, Vipul Sharma1 and Ania Cartier1 1
Department of Biology and Molecular Biology, Montclair State University, 1 Normal Avenue, Montclair, NJ 07043, USA; 2Institut f€ ur Botanik, Technische Universit€at Dresden, Dresden, Germany; *Author for correspondence (e-mail:
[email protected]; phone: +1 973 655 4097; fax: +1 419 791 9834) Received 27 March 2003; accepted in revised form 7 July 2003
Keywords: Arabidopsis suecica, Arabidopsis thaliana, IAA amidohydrolase, IAA conjugate, ILR1, Indole acetic acid, sILR1
Abstract We have recently isolated and characterized a homolog of the Arabidopsis thaliana IAA amidohydrolase ILR1 from Arabidopsis suecica (sILR1). This study examines the enzymatic characteristics of sILR1, as well as spatial and temporal expression of sILR1 compared to ILR1. The sILR1 protein can utilize IAA-alanine and IAA-glycine as substrates more effectively than ILR1. In contrast to ILR1, sILR1 cannot cleave IAAphenylalanine or IAA-leucine as substrates. ILR1 and sILR1 share a pH optimum of 8.0 in Tris buffer. Based on the calculated Kmax value, sILR1 has a higher affinity for IAA-alanine than ILR1. The sILR1 transcript is first detectable in seedlings at day 4 after germination and rises to a steady state level from day 5 to day 15. In A. thaliana, expression of ILR1 begins with a burst at day 1 and decreases over 15 days to a relatively low, but steady state level. Examination of ILR1 and sILR1 transcripts in different tissues shows that both sILR1 and ILR1 are highly expressed in roots, although ILR1 appears more highly expressed in hypocotyls, flowers, and basal leaves than sILR1.
Introduction In higher plants, the hormone indole-3-acetic acid (IAA) is stored conjugated to sugar moieties via an ester linkage or to amino acids or peptides via an amide linkage (Cohen and Bandurski 1982; Bandurski et al. 1995; Walz et al. 2002). Over 95% of the total auxin in a plant can be found in the conjugated form, leaving only a small amount of free hormone available to stimulate cellular growth processes (Hangarter and Good 1981; Campell and Town 1991; Bandurski et al. 1995; Campanella et al. 1996; Lasswell et al. 2000). Amide conjugates account for the bulk of conjugated IAA in dicots studied to date.
IAA-aspartate (IAA-Asp) and IAA-glutamate (IAA-Glu) have been identified as natural conjugates in cucumber (Sonner and Purvis 1985) and soybean (Cohen 1982). IAA-alanine (IAA-Ala) has been detected in Picea abies (Ostin et al. 1992). IAA-Ala, IAA-Asp, IAA-leucine (IAA-Leu), and IAA-Glu have been detected in Arabidopsis thaliana (Barratt et al. 1999; Tam et al. 2000; Kowalczyk and Sandberg 2001). Recently, Walz et al. (2002) suggested that IAA-amino acid conjugates are present in low abundances while IAA bound to peptides and proteins account for the majority of IAA-amide conjugates.
216 IAA amidohydrolases are thought to control the quantity of IAA that is released from the conjugated state into the ‘free’ state (Cohen and Bandurski 1982; Bandurski et al. 1995). Several IAA amidohydrolases have been isolated from A. thaliana (Bartel and Fink 1995; Davies et al. 1999; Lasswell et al. 2000). These genes all fall into a homologous multigene grouping known as the ILR1-like family of genes. Since the ILR1-like family of hydrolases has been well-characterized in A. thaliana but not yet been examined closely in other species (Campanella et al. 2003), we have isolated from Arabidopsis suecica, a close relative of A. thaliana, a homolog of the ILR1 hydrolase, which we have dubbed sILR1 (Campanella et al. 2003). To this end, we are interested in (a) how the IAA amidohydrolases of two closely related species (A. thaliana and A. suecica) have changed in evolution, (b) whether there are biochemical changes in those enzymes, and (c) which regulatory alterations have concomitantly occurred. In this study, we have compared the enzymatic activity and substrate specificity of ILR1 and sILR1. We have also compared the expression of these two homologous genes in different tissues and during seedling development.
Materials and methods Plant materials and plant growth Arabidopsisthaliana (Columbia) seeds were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH, USA) and Arabidopsis suecica seeds from the Sendai Arabidopsis Stock Center (Miyagi University of Education, Sendai, Japan). Seeds used for sources of root, hypocotyl, leaf, and flower tissue were sown in soil (1 : 1 : 1, perlite : sphagnum peat moss : vermiculite) saturated with liquid minimal medium (5 mM potassium nitrate, 2.5 mM potassium phosphate (pH 5.5), 2 mM magnesium sulfate, 2 mM calcium nitrate, 50 M iron-EDTA, 70 M boric acid, 14 M manganese chloride, 0.5 M copper sulfate, 1 M zinc sulfate, 0.2 M sodium molybdenate, 10 M sodium chloride, 0.01 M cobalt chloride). Pots with A. suecica were covered with plastic wrap and
incubated at 4 C for 30 days to induce later flowering. Pots were then transferred to constant light (cool white, fluorescent, 100 mol/s per m2) and grown at 21 C. Plants were slowly hardened off over a 1-week period and fertilized with liquid minimal medium every 2–3 weeks as needed. Roots, hypocotyls and basal leaves were harvested 16 days after germination for A. thaliana and A. suecica. Stems, flowers and apical leaves were harvested at the time point of bolting 57 days after germination for A. thaliana and 129 days for A. suecica. All tissues were stored frozen at 80 C until RNA extraction. For developmental expression studies in seedlings, A. suecica and A. thaliana seeds were germinated in 250-ml flasks with 50 ml of liquid Murashige–Skoog medium (Sigma Corporation). The flasks were agitated at 100 rpm at 23 C in constant light (cool white, fluorescent, 100 mol/s per m2) in a plant growth chamber (Percival Scientific, Model E-30B). Seedlings were collected 1, 2, 3, 4, 5, 10, and 15 days after germination and stored frozen at 80 C until RNA extraction. RNA extraction Total RNA was extracted from 0.2 g of A. suecica or A. thaliana tissue, using the RNeasy RNA extraction kit (Qiagen Corporation). Before extraction, micropestles and all microfuge tubes were treated with an 8% solution of RNA Secure (Ambion Corporation) for 10 min at 65 C. RNA concentration was determined by UV spectrophotometer (Spectronic Genesys 5, Spectronic Corp.) and samples stored at 80 C as separate aliquots until real-time RT-PCR analysis. Real-time RT-PCR This protocol and analysis was based on that of Nakayama and Fujita (1992). Total RNA from various tissues or ages of A. suecica and A. thaliana plants was used for real-time RT-PCR to examine the expression of the sILR1 and ILR1 hydrolase genes. The transcript-specific primers used to amplify sILR1 and ILR1 were 50 -ATTCATGAGAACCCAGAGACA-30 (ILR1F) and 50 CAACCCGAAACCTAACCTCA-30 (ILRR). As an expression control for use in quantitation, universal 18S primers (Ambion Corporation)
217 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. The RT-PCR was combined in a single-tube reaction using 1400 ng of A. thaliana or A. suecica mRNA with components of a One-Step RT-PCR kit (Qiagen Corporation). This single 50-l reaction was carried out in an RNase-free 0.5-ml microfuge tube using a Mastercycler gradient thermocycler (Eppendorf, Inc.). The reverse transcriptase reaction was incubated at 50 C for 1 h, followed by 95 C for 10 min. At the end of the RT reaction, 18S competimer primers (Ambion Corp.) 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 32–42 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-l aliquots were removed from the reaction tubes at the even-numbered cycles, starting at cycle 14, 16, 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-l 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, Inc.) and Scion computer software (Scion, Inc.). Densitometry was performed on each cDNA band by application of the ImageTool Analysis program (University of Texas Health Science Center in San Antonio). Each experiment was repeated two to three times and densitometry values averaged. Growth of bacterial cultures The pEcsILR1 strain (containing the Arabidopsis suecica homolog of ILR1 in the EcoRV site of the pETBlue-2 vector; Campanella et al. 2003) was grown overnight in 5 ml LB medium containing
100 g/ml ampicillin. From this culture 2 ml were transferred to a flask containing 50 ml LB medium including 100 g/ml 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% glucose was included in the medium since the promoter of pETBlue-2 is leaky. Enzyme preparation The bacterial cells were collected by centrifugation for 10 min at 8000 g. The supernatant was removed and the pellet resuspended in lysozyme buffer for cell lysis. The buffer consisted of 30 mM Tris–HCl, pH 8.0, containing 1 mM EDTA, 20% sucrose and 1 mg/ml lysozyme (Sigma). The bacterial pellet was resuspended in 5 ml/initial 50 ml culture 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 l of a 10 mg/ml DNase solution (in 150 mM NaCl, 50% glycerol) was added and the mixture was incubated for 15 min at RT. The extract (100 l volume per assay) was then directly used for the enzyme assay. Enzyme assay The enzyme assay for the conversion of IAA conjugates to free IAA was performed in a 500 l reaction mixture containing 395 l assay buffer, 100 l bacterial enzyme extract and 5 l of a 10 mM stock solution (dissolved in a small volume of ethanol, then diluted with H2O) of each substrate (final concentration 100 M, ethanol concentration was always less then 0.1%). As substrate the amide conjugates IAA-Asp, IAA-Ala, IAALeu, IAA-glycine (IAA-Gly), IAA-phenylalanine (IAA-Phe), IAA-isoleucine, IAA-valine, IBAalanine and the ester conjugates IAA-glucose and IAA-myo-inositol were tested. The assay buffer consisted of 100 mM Tris, pH 8.0, 10 mM MgCl2, 100 M MnCl2, 50 mM KCl, 100 M PMSF, 1 mM DTT and 10% sucrose (Ludwig-Mueller et al. 1996). For the pH-dependence pH values varied between 6.5 and 8.5. For the pH values of 6.5, 7.0 and 7.5 100 mM Hepes buffer were used, for
218 pH 7.5, 8.0 and 8.5 100 mM Tris buffer (LeClere et al. 2002). For the kinetics experiment substrate concentrations of 1, 30, 60, 100, 600 and 1000 M were used (LeClere et al. 2002). The reaction was routinely incubated for 1 h at 40 C. The reaction was stopped by adding 100 l 1 M HCl, and the aqueous phase was then extracted with 600 l of ethyl acetate. The organic phase was removed, evaporated to dryness and resuspended in 100 l methanol for HPLC analysis. For GC–MS analysis the samples were evaporated to dryness, redissolved in 100 l ethyl acetate, methylated with diazomethane (Cohen 1984) and resuspended in ethyl acetate. The experiments were repeated two to four times using different enzyme preparations. All results present means of independent experiments ± S.E. As controls, the uninduced cultures were evaluated. Only those values significantly above the controls were considered as enzymatic activity towards the respective substrate.
with a CP-8400 autosampler (Varian, Walnut Creek, CA, USA). For the analysis 2.5 l of the methylated sample dissolved in 20 l ethyl acetate was injected in the splitless mode (splitter opening 1 : 100 after 1 min) onto a Phenomenex ZB-5 column, 30 m 0.25 mm 0.25 m using He carrier gas at ml min1. Injector temperature was 250 C and the temperature program was 70 C for 1 min, followed by an increase of 20 C min1 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 SIS mode (Varian Manual) was used monitoring ions 130 and 189. Scan rate was 0.6 s scan1, multiplier offset voltage 200 V, emission current 30 A and the trap temperature 200 C. Free IAA enzymatically released was identified according to the retention time on GC compared with an authentic methylated standard and by recording the respective mass spectrum.
HPLC analysis
Results
The total methanol extract (100 l, see above) was subjected to HPLC (Jasco BT 8100 pumps) coupled to an autosampler (Jasco AS-1550), equipped with a 4.6125 mm Lichrosorb C18 5 , reversed-phase column and a Multiwavelength Diode Array detector (Jasco MC-919) set at 280 nm. As solvents, 1% aqueous acetic acid (solvent A) and 100% methanol (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 min1. As software the Borwin chromatography software (JMBS Developments Software for Scientists) was used. Identification of IAA was achieved by comparison with an authentic standard. IAA separated in this system from all conjugates used as substrates with a retention time of 23.4 min (Ludwig-Mueller et al. 1996). The amount of the free IAA (or IBA) released was determined using a standard curve with IAA.
Enzymatic characterization of sILR1
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
Several IAA amino acid conjugates available from Sigma Corp. have been tested using the sILR1 protein from Arabidopsis suecica. In addition, two IAA ester conjugates were tested. The enzyme activity was measured after induction of bacterial cultures with IPTG. Significant amounts of free IAA were released by sILR1 only when IAAalanine or IAA-glycine were used as substrates. The identity of the IAA released was confirmed by GC–MS analysis. Amounts of IAA were calculated after HPLC separation using a standard curve (Ludwig-Mueller et al. 1996). Despite the sequence homology of 98% between ILR1 and sILR1 (Campanella et al. 2003), the substrate specificities for the A. suecica and the A. thaliana enzymes differ. While ILR1 preferentially converts IAA-Leu and IAA-Phe, sILR1 converts IAA-Ala which is also a substrate for ILL2 and IAR3 from A. thaliana (Table 1). IAA-Gly is also a substrate for ILL2, but the conversion is lower than for sILR1. IAA-Gly has not been reported before as an endogenous compound in A. thaliana, and it has yet to be determined whether it is a natural constituent of A. suecica.
219 Table 1. Enzyme activity (nmol IAA released/min per ml).
Substrate
sILR1 (Activity ± S.E.)
ILR1 (as comparison)*
ILL2 (as comparison)*
IAA-Aspartate IAA-Alanine IAA-Phenylalanine IAA-Glycine IAA-Valine IAA-Leucine IAA-Isoleucine IBA-Alanine IAA-glucose IAA-myo-Inositol
Tyr)] between ILR1 and sILR1 occur in the putative peptidase domain. The amino acids at sites 103 and 143 are conserved between IAR3 and sILR1, as well as ILL1, ILL2, ILL3 and ILL5. Based on these amino acid changes, we hypothesized that this observed conservation may indicate that sILR1 is functionally more similar to the
other family-member hydrolases of A. thaliana than to ILR1 (Campanella et al. 2003). We also suggested that the non-conserved amino acid alterations might change the substrate specificity of sILR1 compared to its homolog. Our predictions are now supported in part by the results presented in this study. Although direct comparisons regarding the absolute levels of enzyme activities between ILR1 and sILR1 are not possible due to the fact that we measured sILR1 in crude bacterial extract, our results clearly show that there are differences in substrate specificity for the two enzymes. First, the sILR1 enzyme differs considerably from ILR1: sILR1 has a strong specificity for IAAGly and no activity against IAA-Leu or IAA-Phe (Table 1). Second, the amino acid changes that alter sILR1 to share conserved residues with ILL2 appear to have at least some effect in making the substrate specificity of the two enzymes comparable. Although sILR1 does not share specificity against most of the substrates of ILL2, it does share the specificity for IAA-Gly, which is not found with ILR1 (Table 1). Third, sILR1 has an optimum activity at pH 8.0 which is similar to that found for ILL2 and IAR3, but different from that of ILR1 which has an optimum between 7 and 7.5 (LeClere et al. 2002). Whether the differences in pH optimum and substrate specificity between ILR1 and sILR1 can
221
Figure 4. Relative spatial expression of ILR1/sILR1 transcripts. This histogram is derived from the real-time RT-PCR data in Figure 3. The histogram data are generated by calculating the inverse of the initial cycle at which logarithmic RT-PCR amplification commences.
Figure 3. Spatial expression of ILR1/sILR1 transcripts in two closely related Arabidopsis species. (A) For A. suecica tissues, samples were removed from real-time RT-PCR reactions at 16–40 cycles, electrophoresed on a 1% agarose gel, the gel imaged and each sILR1 product densitometrically analysed. The average densitometric values were determined for two to three experiments and the pixel density plotted against cycle number to show a time course of RT-PCR amplification. (B) For A. thaliana tissues, samples were removed from realtime RT-PCR reactions at 14–42 cycles and ILR1 transcript levels were analyzed as described for sILR1.
be traced back to the residue changes that we have predicted from its primary DNA sequence has yet to be shown in future experiments. Comparison of differences in hydrolase expression Arabidopsis suecica is a convergent species that arose from a hybridization between A. thaliana and Arabidopsis arenosa an estimated 5–10 thousand years ago (O’Kane et al. 1996). In that
relatively short period in evolution, the ILR1 gene in A. suecica has diverged from that of A. thaliana. The divergence of the sILR1’s primary coding sequence has been demonstrated, and our comparison of the spatial and temporal expression of ILR1 and sILR1 have provided additional evidence for an alteration in the regulation of sILR1. It appears that ILR1 expression may be required in A. thaliana at a very early stage of development since we find that the transcript is expressed at high levels starting at day 1 after germination. This pattern of expression generally agrees with data presented on ILR1 expression by Rampey et al. (2002) using ILR1 promoter-GUS-fusion constructs. Rampey et al. (2002) found that the ILR1 promoter was highly active at days 1 and 2 in early A. thaliana seedlings, but GUS expression could no longer be detected after day 3. We observed this same burst of early expression, but the more sensitive system of real-time RT-PCR detection was able to discern low levels of expression up to 15 days after germination. The sILR1 gene expression cannot be detected in A. suecica until several days after germination, and then it is strongly discernible up to day 20 (data not shown). It is possible that, unlike A. thaliana, A. suecica no longer requires expression of this hydrolase as part of the earliest developmental processes. This may be because one of the other homologous members of the ILR1-like family has superceded this function in A. suecica. Alternatively, despite its tight regulation, it may be that the early expression of ILR1 is not an
222 absolute requirement in A. thaliana development and this regulatory alteration in A. suecica does not lead to a delay in maturation. This second hypothesis is supported by the data of Bartel and Fink (1995) that shows that the A. thaliana ilr1 mutant develops normally, despite its insensitivity to exogenous IAA conjugates. The spatial expression of ILR1 and sILR1 reflects both conservation and divergence between A. thaliana and A. suecica. The root remains the major organ of hydrolase expression in both species. Again, Davies et al. (1999) demonstrated that IAR3 is also expressed most highly in the roots. This expression may be a sign for the spatial requirement of the hydrolase enzyme to release free IAA for actively growing root tissues. Further examination of the spatial expression of other ILR1-like family members in A. thaliana and additional species is required for further conjecture. There are several tissues that demonstrate differences in spatial expression between ILR1 and sILR1 including flowers, apical leaves, basal leaves, and hypocotyls. ILR1 is more highly expressed in all these tissues than sILR1. In fact, sILR1 is expressed at the lowest level of any tissues in apical leaves while this is not the case for ILR1. The primers used in the real-time RT-PCR analysis were specific for the sILR1 and ILR1 transcripts. Campanella et al. (2003) demonstrated that these primers clearly amplify only a single cDNA fragment for both A. suecica and A. thaliana. Sequence analysis of the amplified fragment from A. suecica indicates only sILR1 sequence present (data not shown). Additionally, the ILR1-like family members have 51–86% similarity to ILR1 in their cDNA sequences (Bartel and Fink 1995; Davies et al. 1999; Lasswell et al. 2000; LeClere et al. 2002), but these homologs are not amplified by the primers used for ILR1 amplification. It seems unlikely that the A. suecica orthologs, other than the highly homologous sILR1 at 98% similarity, could be amplified by the same primers. Clearly, there has been a divergence between the two IAA amidohydrolases from the two species in terms of their regulation in both a spatial and temporal fashion. Future experiments aim to isolate the regulatory sequences that control the expression of sILR1. These sequences will be compared to those controlling ILR1 to determine how they differ and what the basis may be for the
differences in regulatory control between the two species. We believe that further analysis of this system will give us better insights into the molecular evolution of the ILR1-like gene family and how they changed in the genomes of closely related plant species. This is an ideal system to investigate both the physiological and phylogenomic aspects of evolution. Acknowledgements We thank Lisa Campanella for her great help in editing. IAA-glucose and IAA-myo-inositol were gifts from Dr Jerry D. Cohen. The technical assistance of Mrs. S. Heinze is gratefully acknowledged. This work was supported in part by a DFG grant to J. Ludwig-Mueller. This work was also supported in part by a Sokol grant for undergraduate research from Montclair State University.
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