Structural, Biochemical and Phylogenetic Analyses Suggest that Indole-3-acetic Acid Methyltransferase Is An Evolutionarily Ancient Member of the SABATH Family 1

The plant SABATH protein family encompasses a group of related small molecule methyltransferases (MTs) that catalyze the S -adenosyl-L-methionine dependent methylation of natural chemicals encompassing widely divergent structures. Indole-3-acetic acid (IAA) methyltransferase (IAMT) is a member of the SABATH family that modulates IAA homeostasis in plant tissues through the methylation of IAA's free carboxyl group. The crystal structure of Arabidopsis thaliana IAMT (AtIAMT1) was determined and refined to 2.75 Å resolution. The overall tertiary and quaternary structures closely resemble the two-domain bi-lobed monomer and the dimeric arrangement, respectively, previously observed for the related salicylic acid (SA) carboxyl methyltransferase from Clarkia breweri (CbSAMT). To further our understanding of the biological function and evolution of SABATHs, especially of IAMT, we analyzed the SABATH gene family in the rice ( Oryza sativa ) genome. Forty one OsSABATH genes were identified. Expression analysis showed that more than half of the OsSABATH genes were transcribed in one or multiple organs. The OsSABATH gene most similar to AtIAMT1 is OsSABATH4 . E. coli -expressed OsSABATH4 protein displayed the highest level of catalytic activity towards IAA, and was therefore named OsIAMT1. OsIAMT1 exhibited kinetic properties similar to AtIAMT1 and poplar IAMT (PtIAMT1). Structural modeling of OsIAMT1 and PtIAMT1 using the experimentally determined structure of AtIAMT1 reported here as a template revealed conserved structural features of IAMTs within the active site cavity that are divergent from functionally distinct members of the SABATH family such as CbSAMT. Phylogenetic analysis revealed that IAMTs from Arabidopsis, rice and poplar form a monophyletic N-methyltransferase involved alkaloids synthesis


Introduction
A group of structurally and phylogenetically related methyltransferases (MTs) called the SABATH family was recently identified in plants (D'Auria et al., 2003). Biochemically characterized members of the SABATH family methylate the nitrogen atom or carboxyl groups found in a variety of plant small molecules. Salicylic acid (SA) MT (SAMT), the first characterized member of the family, catalyzes the formation of methyl salicylate (MeSA) from SA and S-adenosyl-L-methionine (SAM) (Ross et al., 1999;Negre et al., 2002). Benzoic acid (BA) MT (BAMT) uses BA to produce methyl benzoate (MeBA) (Murfitt et al., 2000). Some SABATH proteins possess dual functionality exhibiting both SAMT and BAMT activities and are therefore called BSMTs (Chen et al., 2003;Pott et al., 2004). Both MeSA and MeBA are components of the floral scents of some plants, and are also produced by vegetative parts of plants in response to environmental challenge (Chen et al., 2003).
Arabidopsis FAMT (AtFAMT) was identified using high-throughput biochemical assays (Yang et al., 2006). Gene expression analysis suggests that the AtFAMT gene, like other members of the SABATH gene family, has an as yet unidentified role in plant defense (Yang et al., 2006). Indole-3-acetic acid (IAA) MT (IAMT) catalyzes the methylation of the free carboxyl end of the plant hormone IAA. IAMT has been characterized in 6 al., 2005). More recently, gibberellic acid methyltransferases (GAMTs) were shown to specifically methylate several types of gibberellins (Varbanova et al., 2007).
Sequence analysis showed that all these genes belong to the SABATH family (Yoneyama et al., 2006).
The continued identification and biochemical characterization of SABATH proteins will greatly benefit from the identification of the structural features responsible for substrate recognition that must have undergone evolutionary variation to refine their selectivity towards specific small molecules critical to plant development and survival in a variety of ecological niches. To date, the three-dimensional crystal structure of three 7 IAMT (Zubieta et al., 2003;Qin et al., 2005) and FAMT (Yang et al., 2006). These analyses provided useful insights into the structural basis for natural variations of substrate specificity among SABATH proteins. Nonetheless, to more accurately identify the structural determinants responsible for substrate specificity among SABATH proteins, an experimentally determined three-dimensional structure for each member of the SABATH family is necessary, particularly when subtle structural rearrangements or insertions and deletions occur within the core primary sequence of the growing family of SABATH enzymes. This task is particularly daunting as a large number of SABATH sequences have been found in many plant genomes through genomic and EST sequencing projects (D'Auria et al. 2003;F. Chen, unpublished). Comparative genomic analysis of SABATH genes in these plant species, in particular the identification of orthologous genes and the determination of the substrate specificity of the enzymes they encode, will also aid in providing useful insight into the evolution of SABATH proteins within and among plant species and the physiological relevance of small molecule methylation reactions in plant growth and development.
Here, we report the determination of the three-dimensional structure of the Arabidopsis IAMT, the biochemical analysis of IAMT from rice, as well as the phylogenetic analysis of the entire SABATH family from rice and Arabidopsis. The three-dimensional structure of Arabidopsis IAMT was further used to model the active site of rice IAMT as well as of poplar IAMT, whose biochemical activity has recently been demonstrated (Zhao et al., 2007).
The SAM binding C-terminal domain possesses the commonly observed α/β fold of a large superfamily of SAM-dependent MTs that diverge in the number and size of accessory domains (Fig. 1).
The quaternary structure of AtIAMT1 (one homodimer observed per asymmetric unit, Fig. 1) again is shared with CbSAMT (Zubieta et al., 2003). The surface area buried in the dimerization interface is about 1025 Å 2 , which represents only 6.3% of the total surface area of each monomer. As in CbSAMT, and unlike most of the plant small molecule O-methyltransferases structurally characterized to date (Zubieta et al., 2001;Zubieta et al., 2002), the dyad-related monomer of IAMT does not contribute to the active site of its partner molecule (Fig. 1).
The first 49 residues of AtIAMT1 in this particular set of crystal structures form a mobile loop. In fact, recognizable electron density attributable to residues 1-24 is noticeably absent from the refined AtIAMT1 crystal structure which also lacks a bound IAA substrate. The mobile active site capping loop in CbSAMT equivalent to residues 1-24 in AtIAMT1 closes the active site, forming a series of inter-atomic interactions with the carboxyl group of the bound salicylate substrate (Zubieta et al., 2003).

Active Site Topology of AtIAMT1
As expected from sequence alignments, the residues of AtIAMT1 likely to interact with the carboxyl moiety of the IAA substrate are strictly conserved with respect to CbSAMT.
These residues include Lys10, Gln25 (not observed in the electron density and located on the mobile N-terminal active site capping loop) and Trp162 (Trp151 in CbSAMT). The majority of the carboxyl-bearing substrate binding pocket in AtIAMT1 is noticeably hydrophobic and rich in aromatic residues as previously seen in CbSAMT (Fig. 4).

Identification of the SABATH Gene Family in Rice
To identify the complete SABATH gene family from the fully sequenced rice genome (International Rice Genome Sequencing Project, 2005), the protein sequence of CbSAMT was initially used to search the genome sequence database of rice using the BlastP algorithm (Altschul et al., 1990 used reiteratively to search the same sequence database. Through this iterative sequence search, forty-one sequences encoding proteins bearing significant similarity to known SABATH proteins were identified in the rice genome (Supplemental Table SI). It should be noted that among the 41 OsSABATH sequences, fifteen of them appear to encode proteins shorter than 300 amino acid residues. Some of the shorter proteins may be due to inaccurate annotation (Rouze et al., 1999), and some of them may represent pseudogenes.
Some of the genes annotated to encode proteins over 350 amino acid residues in length may also be pseudogenes, as shown in supplemental Figure S1. Additional efforts to characterize these genes will clarify whether individual OsSABATH sequences code for intact genes or pseudogenes.
Mapping the physical locations of the 41 OsSABATH genes revealed that these genes are scattered on seven chromosomes that include chromosomes 1, 2, 4, 5, 6, 10 and 11 (Fig. 5). More than half of the OsSABATH genes (22) are localized on chromosome 6.
In contrast, chromosomes 3, 5 and 10 each contain only one SABATH gene. Twenty two OsSABATH genes are localized in six clusters in which OsSABATH genes are adjacent or separated by one unrelated gene. Cluster C6 contains eight OsSABATH genes (Fig. 5).

Expression analysis of OsSABATH Genes
To obtain information on the biological processes in which OsSABATHs may be involved, comprehensive gene expression analyses using semi-quantitative RT-PCR were performed for all OsSABATH genes using gene-specific primers. PCR employing rice genomic DNA as template was conducted to confirm the effectiveness of the primers used in RT-PCR. Gene expression analyses were performed with leaves, roots and stems from one-month old seedlings, panicles from four month-old flowering plants and germinating seeds.
In these experiments, results for different genes in the same organ are directly comparable, because an identical aliquot of cDNA from the original RT reaction was used in each PCR. To determine whether equal amounts of cDNA were used in the reactions involving different organs, we also performed RT-PCR with primers designed to detect actin mRNA. After RT-PCR, amplified fragments from mRNAs of 23 of the 41 OsSABATH genes were obtained from at least one organ (Fig. 6). The expression of 20 genes was detected in roots, 13 genes in stems, 16 genes in leaves, 16 genes in panicles and 10 genes in germinating seeds. Nine genes showed expression in all tissues examined. In contrast, sixteen genes exhibited no expression in any of the tissues examined.

Identification of OsIAMT1 and Its Biochemical Properties
The rice SABATH gene that is most similar to AtIAMT1 is OsSABATH4. OsSABATH4 encodes a protein spanning 404 amino acid residues with a calculated molecular mass of 43.8 kD. At the amino acid sequence level, OsSABATH4 is 61% identical to AtIAMT1.
To determine whether this gene encodes rice IAMT, full length cDNA of OsSABATH4 was cloned and protein expressed in E. coli. The protein was purified and tested with a group of potential substrates including IAA, indole-3-butyric acid (IBA), salicylic acid, jasmonic acid, farnesoic acid and gibberellic acid. Dichlorophenoxyacetic acid (2,4-D), a synthetic compound structurally highly similar to IAA, was also tested as a substrate.
OsSABATH4 displayed the highest level of catalytic activity with IAA, exhibiting a specific activity of 504±31 pkat/mg protein. The enzyme also displayed activity with IBA and 2,4-D, but only at 2% and 5% levels of the activity measured with IAA respectively (AtIAMT, tested for comparative purposes, possessed 12% and 30% of the activity with IBA and 2,4-D compared with its activity measured using IAA as a substrate, respectively). OsSABATH4 exhibited no activity with salicylic acid, jasmonic acid, farnesoic acid and gibberellic acid used as substrates (AtIAMT also had no activity with these substrates). The substrate specificity of the protein encoded by OsSABATH4 is therefore very similar to AtIAMT. As we have not yet analyzed all rice SABATH proteins and can not rule out the possibility that other OsSABATHs also possess IAMT activity, we named OsSABATH4 OsIAMT1.
To determine the chemical structure of the product of OsIAMT1, the compound produced from the OsIAMT1 enzyme assay with IAA as a substrate was extracted with hexane and analyzed using a GC-MS. As shown in Figure 7, the product showed the same retention time and mass fragmentation spectrum as the authentic methyl indole-3acetate (MeIAA) standard, confirming that OsIAMT1 catalyzes the formation of MeIAA using SAM as a methyl donor and IAA as a methyl acceptor.
To determine the pH optimum of the enzymatic assays, OsIAMT1 was assayed with IAA at buffers with differing pH values between pH 6.5 to pH 10.0. The optimum pH was determined to be pH 7.5. At pH 6.5, the enzyme showed 20% of its maximal activity. At pH 9.0, the activity was 30% of the maximum. As observed for other SABATH proteins that have been biochemically characterized, OsIAMT1 activity can be affected by metal ions. K + , NH 4 + , and Na + all stimulated OsIAMT1 activity by more than respectively, and a kcat of 0.025±0.0001 s -1 .

Molecular Modeling of Rice and Poplar IAMTs
Homology models of OsIAMT1 ( reported here (PDB ID 3B5I). These models show a high degree of similarity of the overall structure among AtIAMT1, OsIAMT1 and PtIAMT1 (Fig. 8), which is a predictable consequence of the protein sequence similarity. Furthermore, these models also exhibit the hydrophobic residues that form the substrate binding site previously observed in AtIAMT1 (Phe158, Leu226, Leu242, Phe243, Val326 and Phe364 in AtIAMT1 sequence) (Fig. 4).

Phylogenetic Analysis of SABATHs
Arabidopsis was the first plant species in which the complete SABATH gene family was identified (Chen et al., 2003;D'Auria et al., 2003). To understand the evolutionary relationships among SABATH proteins, a phylogenetic tree containing the entire set of rice and Arabidopsis SABATH proteins and selected SABATH proteins from other plants was constructed (Fig. 9). When only SABATHs from rice are considered, 41 OsSABATHs group into three clades (I, II and III Previously identified SABATH proteins from other plants group in clades II, IV and V (Fig. 9). CbSAMT and SAMTs isolated from snapdragon, Stephanotis floribunda and Nicotiana suaveolens sit in clade II. Snapdragon BAMT and coffee caffeine synthase (CCS1) reside in clade V. Notably, AtIAMT1, OsIAMT1 and PtIAMT1 form a monophyletic group, which is closely related to putative SABATHs identified from gymnosperm species (Fig. 9).

Structural Basis for Substrate Specialization of SABATH Proteins
The previously determined CbSAMT structure was obtained with SAH (demethylated SAM) and salicylate bound in the active site. The AtIAMT1 structure obtained and described in this report has only SAH and no visible IAA bound to its active site. current structure of AtIAMT1, while the same loop is well ordered in the previously published CbSAMT structure, suggests that this polypeptide segment acts as a dynamic lid to lock down substrates and desolvate the carboxyl group undergoing methylation. This hypothesis is supported not only by the apparent mobility of the capping loop which allows substrate entry but also by the absolute conservation of the capping loop residues interacting with the substrate's carboxyl moiety including Lys10 and Gln25. Through hydrogen bonding interactions, these residues ensure that water molecules solvating the carboxyl group and reducing its reactivity are eliminated. This desolvation mechanism is a prerequisite for enhancing the intrinsic reactivity of the negatively charged carboxyl oxygens now abutting the electrophilic methyl group of bound SAM. Moreover, 42% of the residues located on the mobile loop (1-24) are strictly conserved among the four MTs shown in Figure 4.
Interestingly, standard sequence alignments and homology-based models calculated from the previously published CbSAMT structure predict that Trp226 of CbSAMT is replaced by Gly244 in AtIAMT1, leading to an intuitively simple explanation for the IAA specificity of IAMT1 (Zubieta et al., 2003). However, as a warning against over-reliance on homology models even for proteins possessing a high degree of sequence identity/similarity, it was observed in the experimentally determined AtIAMT1 crystallographic structure that loop α5 is shifted by one residue compared to the sequence alignments, thus superimposing Phe243 of AtIAMT1 with Trp226 of CbSAMT. This significant readjustment of the actual structure relative to the previously published homology model (Zubieta et al., 2003) leaves little room in this portion of the active site compared to CbSAMT. The more restricted AtIAMT1 active site relative to the original homology model based on CbSAMT suggests that in AtIAMT1 the IAA substrate will assume a quite different conformation to avoid a clash of the indole ring of IAA with Phe243.
In order to build a model of IAA bound to AtIAMT1 that takes into account the differences between the IAMT and SAMT active sites noted here, in silico docking techniques were used. The first attempts to computationally calculate a binding orientation for IAA without spatial restraints failed. This lack of initial success is likely due to the absence of the critical N-terminal active site capping loop responsible for highly specific interactions with the carboxyl moiety of bound substrates. A second computational attempt was undertaken, this time using hard constraints to ensure one oxygen of the carboxyl group is located within the appropriate distance for methyl Based on these computational results, Phe158, Pro303 and Ser322 appear to be primarily responsible for the IAA specificity of AtIAMT1 with Ser322 possibly involved in forming a specific hydrogen bond with the indole ring nitrogen of IAA (upon rotation with respect to Cα-Cβ bond, Oγ atom of Ser322 resides as close as 2.9 Å from the indole nitrogen atom). Indeed, all three residues contribute to the IAA binding pocket, and, based on the structure-based sequence alignments, are conserved residues in the three IAMTs listed and are consistently different in SAMTs (Figs. 3 and 4). OsIAMT1 also exhibited expression in multiple tissues. OsIAMT1 showed high levels of expression in roots and panicles (Fig. 6). In rice, IAA plays critical roles in root development, including the elongation of the primary roots, the initiation and elongation of lateral roots, the formation of adventitious roots and root gravitropism (Chhun et al., 2003). Although the mode of action in these cases is not well understood, the concentration of IAA appears to be important in regulating IAA activities in rice roots   Wang et al., 2003). The presence of OsIAMT1 transcripts in rice roots suggest that OsIAMT1 is involved in root development by regulating the homeostasis of IAA in the tissue. IAA also appears to play a role in rice grain development. During grain filling, a dramatic reduction in the levels of IAA in panicles has been observed (Yang et al., 2000).
The high level of OsIAMT1 transcripts in rice panicles (Fig. 6) implies that OsIAMT1 is involved in the reduced level of IAA found there. Expression of IAMTs in leaves of different plant species suggests that their function in leaf development may be evolutionarily conserved. Their divergent expression patterns in certain tissues, for instance, high expression levels in poplar stems and very low expression levels in rice stems, imply that IAMTs may also have acquired lineage-specific roles in different plant species.

Evolution of the SABATH Gene Family
Rice is the second plant species, after Arabidopsis, in which the entire SABATH gene family has been identified. The size of the OsSABATH family, 41 members, is larger than that of the AtSABATH family, which contains 24 members (D'Auria et al., 2003). In contrast to AtSABATH genes which are localized on all five chromosomes, the 41 The majority of OsSABATH genes were transcribed under normal growing conditions (Fig. 6). Some of these genes, such as OsSABATH29, appear to have roles in the general biology of rice plants, as they are expressed in all tissues examined. Other OsSABATH genes, such as OsSABATH19, may have a tissue-specific role, as they are expressed only in some tissues. The expression analysis of OsSABATH genes in leaves, roots and stems presented here was done with tissues from one-month-old plants. It will be interesting to examine the developmental regulation of expression of these genes in the various tissues. The genes that showed no expression in any of the tissues examined may be expressed under stress conditions. In addition, some of the expressed genes may Among all OsSABATHs and AtSABATHs, OsIAMT1 is most related to AtIAMT1, implying that they are likely orthologous genes. In contrast, whether the rice genome encodes AtSABATH proteins with the same catalytic activity as AtJMT, AtBSMT and AtFAMT, respectively, is difficult to predict from this phylogenetic analysis because each of them is more related to other AtSABATHs than to any OsSABATHs (Fig. 9).
Identification and characterization of SABATH genes from related plant species will help determine whether AtJMT, AtBSMT and AtFAMT evolved after the divergence of Arabidopsis and rice lineages. For example, a recent study showed that OsSABATH3 has BSMT activity in vitro (Koo et al., 2007). The phylogenetic placement of OsSABTH3 in the rice-specific clade I, AtBSMT in clade V, and other known SAMTs in clade II ( Fig.   9) suggests that SAMTs emerged several times during the course of SABATH gene evolution. It is interesting to note that caffeine biosynthetic pathways seem to have evolved independently several times in plants as well. This conclusion was drawn based on the observation that the sequence identity between theobromine synthase and caffeine synthase within the genus of Camellia is very high, while the sequence identity among the functionally orthologous N-methyltransferases between Camellia and Theobroma is lower (Yoneyama et al., 2006).
As previously demonstrated, the emergence of a novel SABATH MT activity can occur rapidly, and small changes in primary protein sequences can lead, as for other enzymes of specialized metabolism, to the functional emergence of SABATH proteins with altered substrate preferences (Zubieta et al., 2003;Pichersky et al, 2006). This observation poses difficulties for the functional assessment of SABATH proteins based only on overall sequence similarity to a biochemically characterized protein.
Identification of conserved IAMTs from rice, Arabidopsis and poplar, however, implies A full-length cDNA of AtIAMT1 was cloned into the pHIS8 expression vector (Jez et al., 2000). N-terminal His8-tagged protein was expressed in E. coli BL21 (DE3) cells.
The histidine tag was removed using thrombin protease digestion, and the cleaved protein was purified to greater than 99 % homogeneity by gel filtration chromatography on a Superdex-S200 (Amersham Biosciences, Inc.) FPLC column, equilibrated in 500 mM KCl, 25 mM Hepes-Na + , pH 7.5, 2 mM DTT. AtIAMT1-containing fractions were combined and concentrated to 33 mg/mL. An in-house crystallization screen was used to (v/v) ethylene glycol added to the mother liquor noted above for crystal growth. A 2.75 Å resolution dataset was collected at the National Synchrotron Light Source (NSLS). Data reduction was performed with the XDS program (Kabsch, 1993). The space group was P21 (a=67.3 Å, b=129 Å, c=68.3 Å, b=112.3°) with two molecules per asymmetric unit (solvent content of 63%). The AtIAMT1 structure was determined by molecular replacement using Molrep (Vagin and Teplyakov, 1997) and Clarkia breweri CbSAMT (PDB code: 1M6E, 34 % sequence identity) as a template. A first structure solution, including 345 residues out of the 374 residues of AtIAMT1, was built manually into the experimental electron density maps displayed with Coot (Emsley and Cowtan, 2004) and refined with CNS (Brunger et al., 1998) and Refmac5 (Murshudov et al., 1997). Clear electron density was observed in the active site and interpreted as a SAH molecule.
Despite several attempts at soaking IAA into existing crystals or growing crystals in the presence of IAA, no IAA was observed in the active site.

In silico Docking Experiments
In silico docking experiments were carried out with the software package Schrödinger All programs were run on a bi-Opteron TM 64b Linux computer. Each docking experiment was carried out over an extended area, exceeding the volume of the active site.

Sequence Retrieval and Analysis
The protein sequence of CbSAMT (accession number AF133053) was used initially as a query sequence to search against the translated rice genome database (http://www.tigr.org/tdb/e2k1/osa1) using the BlastP algorithm (Altschul et al., 1990).
Newly identified SABATH-like sequences were used reiteratively to search the same sequence database. The cutoff e-value was set to e -6 . The chromosome locations of the rice SABATH genes were generated by Map Viewer (http://www.ncbi.nlm.nih.gov/mapview/ static/MVstart.html). Phylogenetic trees were produced using PAUP4.0 based on multiple sequence alignments made with ClustalX (Thompson et al., 1997) and viewed using the TreeView software (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).

Gene Expression Analysis via RT-PCR
Semi-quantitative RT-PCR expression analysis of rice SABATH genes were performed as previously described (Chen et al., 2003). Primer sequences are shown in the Supplemental Table SII. Total RNA was isolated with Trizol (Invitrogen, Carlsbad, CA).
1.0 µ g total RNA was synthesized into first strand cDNA in a 20 µ L reaction volume using the iScript cDNA synthesis kit (Bio-rad Laboratories). 1.0 µ L of the resulting cDNA mixture was used in each PCR using the following conditions: initial denaturation at 95°C for 2 min followed by 30 cycles at 95°C for 45 sec, 54 °C for 45 sec and 72 °C for 60 sec, and then followed by a final extension step at 72 °C for 10 min.

Purification of Recombinant OsIAMT1
OsIAMT1 full-length cDNA was amplified from rice root tissues by RT-PCR using the forward primer 5'-CACCATGGCTCCTAAAGGTGACAATGTTG-3' and the reverse primer 5'-CTACTACTATGCGGATGCTGCTATAC -3'. The resulting PCR product was cloned into the pET100/D-TOPO vector (Invitrogen, Carlsband, CA). The construct was transformed into E. coli strain BL21 Codon Plus (Invitrogen, Carlsband, CA). Protein expression was induced by IPTG for 18 h at room temperature. His-tagged OsIAMT1 protein was purified from E. coli cell lysate using Ni-NTA agarose following manufacturer's instructions (Invitrogen, Carlsband, CA). Protein purity was verified by SDS-PAGE and protein concentrations were determined using the Bradford assay.

Radiochemical MT Activity Assay
Radiochemical MT assays were performed in a 50 were performed for each compound. In addition to its use in determination of substrate specificity of OsIAMT1, the radioactive MT assay was also used to determine kinetic parameters, pH optimum and effector effects of OsIAMT1.

Determination of Kinetic Parameters of OsIAMT1
In all kinetic analyses, the appropriate enzyme concentrations of OsIAMT1 and incubation times were chosen so that the reaction velocity was linear during the reaction was varied. Km values and maximum velocity values were obtained as previously described (Chen et al., 2003). Final values were an average of three independent measurements after non-linear regression analyses using the Michaelis-Menten equation.
pH Optimum for OsIAMT1 Activity OsIAMT1 activity was determined in a 50 mM Bis-Tris propane buffer for the pH range 6.5 to 10 using the standard IAMT assay described above. The resultant kinetic constants used for determination of the pH optimum were an average of three independent assays.

Effectors
To examine effects of metal ions on OsIAMT1 activity, standard IAMT assays were performed in the independent presence of each of the following salts at 5 mM final concentration: KCl, CaCl 2 , NH 4 Cl, NaCl, MgCl 2 , MnCl 2 , CuCl 2 , FeCl 2 and ZnCl 2 .
Results presented were an average of three independent assays.

Product Identification
A reaction containing 150 µ g of purified OsIAMT1, 1 mM IAA and 600 µ M SAM was incubated in a 1 ml reaction volume containing 50 mM Tris-HCl, pH 7.5, for 4 h at 25 °C. The product was extracted with 1.5 ml hexane, the hexane layer concentrated under N 2 gas, and the resultant organic concentrate analyzed on a Shimadzu GC (GC-17A)-MS (QP 5050A) system. A DB-5 column (30 m by 0.25id by 0.25 µm) was used with helium as carrier gas at a flow rate of 1 ml/min. As a control, a similar reaction was performed, except that OsIAMT1 protein was denatured by boiling at 100 o C for 10 min before addition to the assay. A MeIAA authentic standard was dissolved in ethanol, and a volume containing 1µg MeIAA was injected into the GC-MS in a split (1/30) mode. The GC program was as follows: 2 min at 80 °C, ramp to 300 °C at 8 °C per min followed by a 5 min hold at 300 °C. The compound was identified by comparison of GC retention times and mass spectra with those of the authentic standard.

Homology-based Structure Modeling
Based on the structure of AtIAMT1, homology models of OsIAMT1 and PtIAMT1 were calculated. First, a sequence alignment with AtIAMT1 was performed with Blast     indicate conserved residues, white characters in red boxes indicate strict identity and red characters in white boxes indicate similarity. The secondary structure elements indicated above the alignment are those of AtIAMT1, whose structure has been experimentally determined and described here. Residues indicated with "&" below the alignment are SAM/SAH binding residues. Residues indicated with "*" are residues that interact with the carboxyl moiety of indole-3-acetate. Residues indicated with "#" interact with the aromatic moiety of the substrate, and are important for the substrate selectivity. CbSAMT is Clarkia breweri SAMT (Ross et al., 1999). PtIAMT1 is poplar IAMT (Zhao et al., 2007). OsIAMT1 is rice IAMT (this study). This figure was prepared with ESPript (Gouet et al., 1999). localized on seven chromosomes (1, 2, 4, 5, 6, 10 and 11). Twenty two genes are situated in six clusters (C1 to C6). In each cluster, neighboring genes are found either as tandem repeats or separated by one gene that is not a SABATH family member.