AUXIN CONJUGATE HYDROLYSIS DURING PLANT-MICROBE INTERACTION AND EVOLUTION J. Ludwig-Müller 1, A. Schuller 1, A.F. Olajide 2, V. Bakllamaja 2, J.J. Campanella 2 ABSTRACT Plants regulate auxin balance through de novo synthesis, degradation, transport and conjugation. Several genes encoding auxin conjugate hydrolases were isolated from the dicots Arabidopsis suecica (a close relative to Arabidopsis thaliana), Brassica rapa (a more distantly related species from the Brassicaceae), Medicago truncatula (a model legume), the monocot Triticum aestivum and the gymnosperm Pinus taeda. Expression of the hydrolase genes in E. coli allowed the biochemical characterization of substrate specificity which clearly differed among the amidohydrolases. These investigations will increase our understanding how auxin homeostasis has evolved. Also, different temporal and spatial expression patterns for the hydrolase genes from various species were examined. In addition, auxin conjugate hydrolases might play a role in the establishment of pathogenic or symbiotic interactions with microorganisms. 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. 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 (for a recent review on auxin conjugates see Seidel et al., 2006). IAA amidohydrolases are thought to control the quantity of IAA that is released from the conjugated state into the free, active state (Cohen and Bandurski, 1982). Several IAA amidohydrolases have been isolated from Arabidopsis thaliana (Bartel and Fink, 1995; Davies et al., 1999). Since the ILR1-like family of hydrolases has been wellcharacterized in Arabidopsis thaliana but not yet been examined closely in other species, we have isolated from several other species amidohydrolase homologues and studied their biochemical properties, as well as their expression during development and interaction of host plants with microorganisms. To this end, we are interested in 1) how the IAA amidohydrolases of closely and distantly related species have changed in evolution, 2) whether there are biochemical changes in those enzymes, and 3) which regulatory alterations have concomitantly occurred. MATERIALS AND METHODS Plant material. Different tissues from the following plants were employed in this analysis; the dicots Arabidopsis suecica, Brassica rapa, and Medicago truncatula, the monocot Triticum aestivum and the gymnosperm Pinus taeda (Table 2). 1 Institut für Botanik, Technische Universität, 01062 Dresden, Germany 2 Montclair State University, Montclair, NJ 07043, USA 34
cdna cloning and expression. The cdna sequences for the T. aestivum, M. truncatula and P. taeda hydrolases were identified in The Institute for Genomic Research (TIGR) sequence database and cloned using PCR methods (Campanella et al., 2003; 2004). The A. suecica hydrolase was amplified directly from cdna using degenerate primers homologous to A. thaliana amidohydrolases (Campanella et al., 2003). Finally, the B. rapa hydrolases were PCRamplified with degenerate primers and full length clones were isolated by RACE (Schuller and Ludwig-Müller, 2006). The resulting amplified cdnas were ligated into E. coli expression vectors and the correct orientation and frame was confirmed by sequencing the construct. The protein was expressed after induction of transformed cells with 1 mm IPTG (e.g. Campanella et al., 2004) and the enzyme assay was performed with a crude protein preparation. 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 ca 2.5 mg total protein) and 5 µl of a 10 mm stock solution (dissolved in a small volume of ethanol, then diluted with H 2 O) of each IAA-conjugate substrate (final concentration 100 µm). The different substrates used in this study are shown in Table 1. Analysis of the reaction product was carried out by HPLC. The amount of the free auxin released was determined using a standard curve for the respective standard substance. Real-Time RT-PCR. Total RNA was extracted from ~0.2 g of plant tissue, using the RNeasy RNA extraction kit (Qiagen Corporation) and was used for real-time RT-PCR to examine the expression of the different hydrolase genes under various conditions (Table 2). Analyses were performed on two to four biological replicates for each treatment. RESULTS AND DISCUSSION Biochemical characterization and phylogenetic relationship. So far we have analyzed a range of auxin conjugate hydrolases from different species including three dicot, one monocot and one gymnosperm species. The characterization of the enzyme activity expressed in E. coli together with sequence comparison revealed that the phylogenetic relation of the hydrolase genes does not allow prediction as to which substrates might be cleaved by the respective enzyme (Fig. 1, Table 1). It rather seems that small sequence changes in either the putative hydrolase domain or other domains important for enzymatic activity can lead to a different substrate preference. In general, there are five distinct branches of hydrolases that can be distinguished (Fig. 1A): i) the ILR1 branch consisting of the ILR1 homologs of A. thaliana, A suecica and the moss Physcomitrella patens (J. Ludwig-Müller, unpublished results), ii) the ILL3 branch including A. thaliana ILL3 and M. truncatula IAR33, iii) the ILL6 branch with A. thaliana ILL6, B. rapa ILL6 and M. truncatula IAR36, iv) the ILL1/2 branch with A. thaliana ILL1 and ILL2 and B. rapa ILL2, and v) the IAR3 branch, being the largest group including four of the M. truncatula hydrolases, A. thaliana and B. rapa IAR3 homologues as well as T. aestivum and Oryza sativa IAR3. A. thaliana ILL5 clusters within the IAR branch and P. taeda IAR3 clusters together with members of the ILL6 branch. However, there are differences in the phylogenetic tree (generated with CLUSTALW) if the whole sequence alignment and the alignments of the hydrolase domains are compared (Fig. 1B). Comparison of A. thaliana, A. suecica, B. rapa, and T. aestivum sequences showed close 35
Table 1. Biochemical characterization of auxin conjugate hydrolases from different plant species. The name of the hydrolases are derived from their closest homologue in Arabidopsis thaliana. IAA: indole-3-acetic acid; IBA: indole-3-butyric acid. For IAA conjugates with the amino acids alanine, aspartate, glycine, isoleucine, leucine, phenylalanine, valine and the ester conjugate with glucose were tested, for IBA the amino acid conjugates with alanine and glycine. 1 Minor activites were found with other substrates. *This hydrolase is 5 and 3 truncated ( in comparison to the other hydrolases that have so far been isolated. The Pinus clone is also truncated ( and not full length. It is missing 400 bp from the 5 end which might also affect its activity. Plant species Name of hydrolase Preferred substrates 1 Arabidopsis suecica silr1 IAA-alanine, IAA-glycine Brassica rapa BrILL2 IAA-alanine, IAA-valine BrIAR3 IAA-alanine Medicago truncatula MtIAR31 IAA-aspartate, IBA-alanine MtIAR32 MtIAR33 MtIAR34 ΔMtIAR35* MtIAR36 IAA-aspartate, IBA-alanine IAA-aspartate, IAA-glucose IAA-aspartate, IAA-glucose, IBA-alanine No activity IAA-alanine, IAA-glycine, IAA-isoleucine, IAA-glucose Pinus taeda ΔPtIAR31** IAA-aspartate, IBA-alanine Triticum aestivum TaIAR3 IBA-alanine, IBA-glycine 36
A i ii iii iv v B Fig. 1. Phylogenetic analysis of the hydrolases described in the text. (A) The whole protein sequence was aligned, (B) the protein sequences of the putative hydrolase domains were used for alignment. As: Arabidopsis suecica, At: Arabidopsis thaliana, Br: Brassica rapa, Mt: Medicago truncatula, Os: Oryza sativa, Pp: Physcomitrella patens, Pt: Pinus taeda, Ta: Triticum aestivum. homology between A. thaliana and B. rapa, which also have the same substrate specificity. However, the A. thaliana and A. suecica ILR1 homologs differ quite substantially in their substrate specificity. Sequence comparison indicates that a few amino acid changes in AsILR1 makes it more homologous to the AtIAR3 enzyme (Campanella et al., 2003) with similar enzymatic properties concerning the preferred substrates (AtIAR3 preferentially cleaves IAAalanine, Davies et al., 1999). Finally, the T. aestivum homolog cleaves preferentially IBA conjugates which might be due not only to changes in the hydrolase domain but also in other parts of the enzyme which make it more distantly related in the IAR3 branch. It will be interesting to see whether the close homologue from rice will also cleave IBA conjugates. Within the M. truncatula IAR3 family, there are three groups that arise when amino acid sequences are compared, but only two main groups that become evident when substrate specificity is examined. 37
Important residues outside the putative hydrolase domain were identified so far for the Enterobacter agglomerans hydrolase (Chou et al., 2004). It will be our future goal to identify such residues to study how the hydrolases might have evolved from each other and how their preference for a set of substrates might be relevant for the development of the plant. Expression during development. In Table 2 a compilation of the expression analysis during development and in different tissues is given for the various hydrolases analyzed so far. Most of the hydrolase transcripts are expressed during seedling development and in various tissues indicating overlapping functions. Table 2. Temporal and spatial expression analysis of different hydrolases by Real Time PCR. nd = not determined so far. DAG = Days after germination. Plant species Hydrolase Tissue DAG Arabidopsis suecica silr1 Seedling, root, hypocoyl, basal leaf, apical leaf, stem, flower 4-15 Brassica rapa BrILL2 Root nd BrILL6 Seedlings, root 5 BrIAR3 Root, stem, rosette leaf nd Medicago truncatula MtIAR31 Seedling, root, stem, basal leaf, terminal leaf, flower 1-20 MtIAR32 Seedling, root, stem, basal leaf, terminal leaf, flower 1-20 MtIAR33 Seedling 1-20 MtIAR34 Seedling, root, stem, basal leaf, terminal leaf, 1-20 MtIAR35 Seedling, root, stem, basal leaf, flower 1-20 MtIAR36 Seedling, root, stem, basal leaf, terminal leaf, 1-20 Triticum aestivum TaIAR3 Seedlings, coleoptile, stem, radicle, root, lower and upper leaf 1-20 38
Expression during pathogenesis and symbiosis. Regulation of host-symbiont interaction remains an important interest to those who study both plant and symbiote physiology. The exchange of signals during the initial phases of contact and colonization are especially crucial for the regulation of an intricate relationship between two organisms during symbiosis. Plant hormones may constitute at least part of these signals (Barker and Tagu, 2000). The involvement of auxins in nodule development is feasible, since they are highly organized structures and similar to lateral roots (Hirsch and La Rue, 1997). Levels of auxins were increased in arbuscular mycorrhizal roots (Kaldorf and Ludwig-Müller, 2000). Again, hydrolysis of free auxin from conjugates might play an important role during these processes. On the other hand, auxin is also known to regulate among other signals the gall size during the clubroot disease (Grsic-Rausch et al., 2000). This important disease of Brassicaceae is caused by the obligate biotrophic pathogen Plasmodiophora brassicae and leads to tumorous swellings of the root (Ludwig-Müller, 1999). Next to an increase in auxin biosynthesis the hydrolysis from inactive conjugates might be a way to increase endogenous auxin levels. Table 3. Expression analysis of different hydrolases during pathogenesis or symbiosis by Real Time PCR. The nodulation experiment was performed with Sinorhizobium meliloti, the arbuscular mycorrhizal colonization with Glomus intraradices. The clubs were induced after inoculation with the protist Plasmodiophora brassicae. nr = not regulated; -- = downregulation; + = upregulation. Plant species Hydrolase Clubroot Nodulation Arbuscular mycorrhiza Brassica rapa BrILL2 nr BrILL6 -- / + BrIAR3 -- Medicago truncatula MtIAR31 ++ + MtIAR32 + + MtIAR33 ++ +++ MtIAR34 ++++ +++ MtIAR35 +++ +++ MtIAR36 (+) + Comparison of the expression of conjugate hydrolases in different symbiotic and pathogenic systems has shown that 1) conjugate hydrolases are highly and moderately upregulated during nodulation and arbuscular mycorrhizal symbiosis, respectively, and 2) might be involved in a negative regulation of auxin content in the Brassica-clubroot interaction (Table 3) 39
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