Corresponding author details: James J. Campanella, Montclair State University, Dept. of. Biology, 1 Normal Avenue, Montclair, New Jersey USA.

Transcription

1 Plant Physiology Preview. Published on June 29, 2018, as DOI: /pp Campanella et al. 1 1 Short Title: The M20D peptidase MpILR1 of Marchantia polymorpha Corresponding author details: James J. Campanella, Montclair State University, Dept. of Biology, 1 Normal Avenue, Montclair, New Jersey USA Evidence for exaptation of the Marchantia polymorpha M20D peptidase MpILR1 into the tracheophyte auxin regulatory pathway James J. Campanella 1*, Stephanie Kurdach 1, Joy Bochis 2, and John V. Smalley 1,2 1 Montclair State University, Dept. of Biology, 1 Normal Ave, Montclair, New Jersey Bergen Community College, Dept. of Biology and Horticulture, 400 Paramus Rd, Paramus, New Jersey One-sentence summary: Characterization of the ancient auxin conjugate hydrolase MpILR1 from liverwort provides evidence of its exaptation in the evolution of tracheophytes Author Contributions: J.J.C. conceived the project, performed phylogenetic analysis, and wrote the article with the contributions of all the authors; S.K. cloned MpILR1, performed the hydrolysis experiments, and helped with the writing; J.B. performed the calculations for codon usage; J.V.S. conceived of the codon usage analysis, helped analyze data, and assisted with the writing *Corresponding Author james.campanella@montclair.edu 1 Copyright 2018 by the American Society of Plant Biologists

2 Campanella et al Abstract Auxin homeostasis is tightly regulated by several mechanisms, including conjugation of the hormone to specific moieties, such as amino acids or sugar. The inactive phytohormone conjugate is stored in large pools in plants and hydrolyzed to regain full activity. Many conjugate hydrolases (M20D metallopeptidases) have been identified and characterized throughout the plant kingdom. We have traced this regulatory gene family back to liverwort (Marchantia polymorpha), a member of the most ancient extant land plant lineage, which emerged approximately 475 million years ago. We have isolated and characterized a single hydrolase homologue, dubbed M. polymorpha IAA-Leucine Resistant1 (MpILR1) from liverwort. MpILR1 can hydrolyze two auxin (indole acetic acid, IAA) substrates (IAA-Leucine, IPA-Alanine) at very low levels of activity, but it cannot hydrolyze the two native auxin conjugates of liverwort (IAA-Glycine and IAA-Valine). We conclude from these results that liverwort likely does not employ active auxin conjugate hydrolysis as a regulatory mechanism, and that conjugate homeostasis likely takes place in liverwort by passive background degradation. Further, we present evidence that MpILR1 was likely exapted by tracheophytes over evolutionary time into the auxin regulatory pathway. 41 2

3 Campanella et al Introduction Auxin is a ubiquitous phytohormone that can be found throughout the entire plant kingdom from non-vascular plants through gymnosperms to angiosperms. The hormone, most commonly found in the form of indole acetic acid (IAA), is essential in the regulation of growth and development of stems, shoots, leaves, and roots. The concentration of IAA in a plant is critical: low concentrations of IAA positively impact plant physiological processes, but high concentrations are inhibitory and toxic (Bandurski et al., 1995). In addition, precisely minute, spatio-temporally regulated quantities of the phytohormone are required for proper stem, shoot, leaf, and root development. For this reason, auxin metabolism is tightly regulated, by four major processes: biosynthesis, transport, conjugation, and degradation (Normanly and Bartel, 1999; Ljung et al., 2002). One common regulatory system for auxin metabolism is molecular conjugation, in which auxin is stored in conjugated forms that are generally thought to be inactive (Ludwig-Müller 2011). There are two major types of conjugated molecules: an amide-linked form bound to one or more amino acids and an ester-linked form mostly bound to sugar(s). These two types of conjugates are found in varying concentrations among the diverse tissues of bryophytes, sporophytes, gymnosperms, and angiosperms (Sztein et al., 1999, 2000). Up to 90% of auxin in a plant is conjugated into these storage forms (Bandurski et al., 1995; Campanella et al., 1996), although these forms vary by species. Free, active auxin may be made available by hydrolyzing the conjugate molecule from the side chain. The evolution of the auxin conjugation process can be traced back to the earliest terrestrial plants. The most ancient land plants, the bryophytes, have been studied for their auxin metabolism (Sztein et al., 1999, 2000) and all have been found to produce auxin conjugates. 3

4 Campanella et al Most liverwort species (including Marchantia polymorpha, Pallavicinia lyellii, Reboulia hemisphaerica, and Sphaerocarpos texanus) possess an IAA conjugate distribution in which IAA-amide conjugates account for more than half the auxin in the cell (~52%), IAA-ester conjugates, for ~20%, and free IAA, for ~28% (Sztein et al., 1999). In mosses, IAA-amide conjugates dominate the total IAA composition (~85%), with only a minor amount of IAA-ester conjugates (~5%) and free IAA (~10%) (Sztein et al., 1999). However, our understanding of the evolution of the regulatory mechanism for active auxin conjugate hydrolysis is incomplete. Evidence suggests that auxin production and regulation has changed over evolutionary time. Charophytic algae appear to be the direct aquatic ancestor of terrestrial bryophytes (Hori et al., 2014). Charophytes have relatively high levels of free IAA, and conjugation occurs slowly, if at all. These algae employ an IAA biosynthesis and degradation strategy to regulate auxin metabolism (Sztein et al., 1999, 2000). The first terrestrial plants may have evolved few changes from the processes of auxin regulation found in algae (Hori et al., 2014). Sztein et al. (2000) concluded that although bryophytic liverworts contain IAA conjugates, hydrolysis occurs at a very slow rate and may not be an active process as the one seen in tracheophytes. Ludwig-Müller et al. (2009) proposed that auxin conjugation may occur in moss (Physcomitrella patens) as an intermediate in auxin inactivation and that conjugate hydrolase genes evolved in tracheophytes. Further, Ludwig-Müller et al. (2009) suggested that moss may be a dead end for active auxin hydrolysis and that no earlier species possessed this regulatory pathway. Our previous research has demonstrated that auxin conjugate hydrolase homologues are functionally highly conserved among angiosperms to help regulate auxin activity (Campanella et 4

5 Campanella et al al., 2003a). More recent studies demonstrate that the genomes of gymnosperms and even ferns have genes encoding regulatory hydrolases (Campanella et al., 2014). If tracheophytes have putative auxin conjugate hydrolases, then they must have evolved from ancestral species. Motivated by an understanding that highly conserved genes must have an evolutionary source, we were prompted to search back to liverwort (Marchantia polymorpha), a member of the most ancient extant land plant lineage, which emerged approximately 475 million years ago (Wellman et al., 2003). As in any plant, M.polymorpha is dependent upon auxin for growth and development. Eklund et al. (2015) demonstrated that auxin is essential for gametophyte development and promotion of gemma dormancy, and Solly et al. (2017) found that auxin works as a growth-promoting signal in thallus development. Additionally, liverwort is just as sensitive to the toxic effects of high auxin concentrations as other plants; exogenous auxin at low concentrations stimulated thallus growth, but interference with liverwort auxin conjugation caused severe stunting and loss of differentiated cell fates (Solly et al., 2017). We searched the genome of M. polymorpha, employing the newest genomic sequence (v3.1) (Bowman et al., 2017) and were able to identify one ILR1 hydrolase homologue. Here we characterize this liverwort hydrolase for enzymatic activity and discuss its implications for the evolution of the pathway regulating auxin conjugate hydrolase in plantae

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7 Campanella et al Results Gene Structure of MpILR1 in M. polymorpha Differs from That of Hydrolases in Tracheophytes Previous experience with prokaryotic contamination of the first moss genome database (v1.1) (Rensing et al., 2008) sensitized us to the possibility that our liverwort MpILR1 (OAE ) hydrolase might not be a plant gene. However, we were able to confirm its plantae origins in M. polymorpha based on several factors. First, based on a GenBank BLAST search, the DNA sequence of MpILR1 demonstrated an average 38.8% similarity to a spectrum of tracheophyte hydrolases and its protein sequence bore a moderate similarity (average 60%) (Fig. 1). Additionally, we performed two-dimensional principal coordinate analysis of codon usage (Fig. 2) and found that MpILR1 fell squarely amid eukaryotic hydrolases, lying far from prokaryotic or Archaean homologues. Included in this analysis were prokaryotic hydrolases capable of breaking down IAA-Aspartate. The structure of the MpILR1 protein includes the five conserved amino acids that characterize eukaryotic M20D peptidase structures: Cys137, His139, Glu173, His197, and His397 (Rawlings and Barrett, 1995; Bitto et al., 2009) (Fig. 3). These residues are not conserved in bacterial species and indicate the Mn +2 -ion binding site of an amido-auxin-type hydrolase (structural sub-category Cd08017). Further study of the genomic structure of MpILR1 indicates that the gene includes five exons and four introns (Fig. 4). The total length of the genomic sequence is 2554 base pairs. The length of the MpILR1 coding sequence is 1404 base pairs. The study conducted by Davies et al. (1999) and later genomic work indicate that the intronic structure of the tracheophyte M20D family consists of four introns and five exons; thus, it appears that the original liverwort intron/exon structure has been conserved since terrestrial plants first evolved. 7

8 Campanella et al MpILR1 does not possess an endoplasmic reticulum-localization signal at its carboxyl- terminal, such as observed in many angiosperm hydrolases, including those of Arabidopsis thaliana (Bitto et al., 2009) MpILR1 Phylogeny Supports its Position as the Ancestor of all Tracheophyte Auxin Conjugate Hydrolases The phylogenetic analysis performed on the amido-hydrolase orthologues (Fig. 5) suggests that the outgroup M. polymorpha MpILR1 may be the ancestor of all tracheophyte hydrolases that evolved after liverwort. The cladogram employs amino acid sequences and manifests clear phylogeny leading to present-day amido-conjugate hydrolases i.e. the liverwort hydrolase shares a common ancestry with fern hydrolases and eventually gymnosperm and angiosperm hydrolases. This observation is supported by bootstrap values, which are all over 500. A single copy of the putative auxin amidohydrolase homologue is found in liverwort. Additionally, we detected a single homologue in Klebsormidium flaccidum, the direct algal progenitor of liverwort (Hori et al., 2014). Going back further in evolutionary time, a single 8

9 Campanella et al hydrolase homologue, related to that of both algae and liverwort, can be detected in the cyanobacterium Hassallia byssoidea. However, moving forward in time after liverwort, an expansion of the amidohydrolase family has occurred over the last 475 million years since liverwort initially evolved (Table 1). Genetic redundancy of the M20D family can be seen as early as in the ferns with two isoforms, and later in gymnosperms that have anywhere between two to four isoforms (Fig. 1; Table 1). In angiosperms, it becomes even more apparent with the redundant isoforms of amidohydrolases reaching as many as 15 in grape and 12 in soybean. This trend continues in monocots, with 11 paralogues detected in corn. 9

10 Campanella et al Wild-Type MpILR1 has Weak Hydrolytic Activity Against Auxin Conjugates The MpILR1 hydrolase recognized only a few auxin conjugate substrates (IAA-Leu, IPA-Ala) and further had very low activity against those substrates (~10.20 and ~1.66 pmol auxin/min/ml, respectively) (Table 2). These levels of hydrolysis are lower than those of 10

11 Campanella et al tracheophyte auxin conjugate hydrolases that have been characterized, where hundreds or more picomoles of auxin have been observed to be released per minute with the appropriate substrate (Davies et al., 1999; Campanella et al., 2003b, 2004, 2008, 2014). It should be noted that the MpILR1 mrna appears to be expressed in liverwort, since we initially detected its cdna in the liverwort expression database generated by Bowman et al. (2017) The Mutated MpILR1 L244S Enzyme has Both Greater Activity and Recognition Against Auxin Conjugates The low levels of MpILR1 hydrolysis against the most common auxin conjugate substrates suggested that this liverwort enzyme might have a structure unlike that of tracheophyte auxin conjugate hydrolases that later arose during evolution. Prompted by observation of the minimal levels of hydrolysis in the wild-type MpILR1, our next goal was to determine what structural alterations to MpILR1 may have led to the tracheophyte evolution in which the M20D enzyme family recognized and became more active against those substrates. The X-ray crystallographic analysis of AtILL2 by Bitto et al (2009) suggested to us potential structural changes that could have arisen over evolutionary time in MpILR1 to make it the more active enzyme found in tracheophytes. A range of mutant hydrolases were characterized by Bitto et al (2009), where single amino acid changes led to loss of function in 11

12 Campanella et al AtILL2. Only one of these alterations could be detected in the MpILR1sequence to account for its reduced function. The mutation was originally identified by Davies et al (1999) in AtIAR3. The structural alteration is a substitution in which Serine206 is mutated to a leucine. We located the analogous 206 amino acid in MpILR1 (Leucine244) and hypothesized that this wild-type leucine residue may be inhibiting substrate recognition and activity and obstructing the active site. Based on this hypothesis, we constructed a mutant version of MpILR1 in which Leucine244 was changed to the serine conserved in later tracheophytes. The new - MpILR1 L244S construct was transformed into Escherichia coli and expressed for enzymatic analysis. The MpILR1 L244S protein shows both greater substrate recognition and a higher level of hydrolytic activity than the wild-type enzyme (Table 2). The re-engineered, mutant enzyme recognized IBA-Alanine and IAA-Isoleucine, which the wild type did not hydrolyze at all. Hydrolytic activity did increase slightly against IAA-Leucine from ~10.20 to ~16.23 pmol auxin/min/ml hydrolyzed (Table 2), while hydrolysis against IPA-Alanine seemed to increase by 12

13 Campanella et al ~11-fold. We still observed no hydrolysis of the native auxin conjugates (IAA-Val and IAA-Gly) of liverwort. It is unlikely that MpILR1 L244S was more active than the wild-type MpILR1 because of differences in protein concentration. We performed a PAGE control to determine if L233S and MpILR1 were being expressed at different levels. They appeared to be alike in terms of expression when examined at 52 kd of molecular weight in the PAGE analysis (data not shown)

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15 Campanella et al Discussion MpILR1 and Evolution in Plantae Auxin conjugate amidohydrolases create an evolutionary paradox i.e. if bryophytes do not have active hydrolytic regulation of auxin conjugates, then where did these hydrolases arise? Exaptation (Gould and Vrba, 1982) could explain the evolutionary source of these enzymes given the evidence that we present here. Exaptation can be defined as the evolutionary co-opting and strengthening of one trait for a different purpose or role. Liverwort may provide one of the earliest examples of an enzyme that would eventually be exapted for use in tracheophytes. MpILR1 is an inefficient and low-functioning hydrolase with standard auxin conjugates tested as substrates (Table 2), compared to tracheophyte hydrolases. Additionally, MpILR1 does not appear to hydrolyze either of the two auxin conjugates found in M. polymorpha: IAA-Valine and IAA-Glycine (Sztein at al., 1999) (Table 2). We conclude from these two observations that MpILR1 may have an unknown substrate and function entirely different from what we see later in tracheophytes. The initial role of MpILR1 may have been unconnected with the active regulation of auxin. However, through a series of mutations and alterations over evolutionary time, MpILR1 became exapted into the auxin regulatory pathway because it had the potential for a different purpose/function. We note that Sztein et al. (1999) do caution that their identification of individual auxin conjugates was tentative in bryophytes since they used thin layer chromatography separation after incubation with IAA and not mass spectrometry. It is still possible that MpILR1 contributes to M. polymorpha physiology by hydrolyzing IAA-Leucine and IAA-Alanine at low levels, and this possibility will have to be tested in future experiments. 15

16 Campanella et al Although the active site of MpILR1 has a moderate similarity (57 60 %) to corresponding sequences in modern plantae, it is difficult to identify which of many structural alterations from evolutionary time were necessary to modify it into the enzyme we recognize now in tracheophytes. We have characterized at least one structural change that was clearly necessary, but not sufficient, for the full activity in tracheophytes. The conserved Serine206 (Bitto et al., 2009) that is found in the active site of M20D hydrolases is not present in MpILR1, where it is replaced at the corresponding position with a Leucine244. Bitto et al. (2009) suggested that this substitution results in a loss of hydrogen bonds to His380 and Asn337. Additionally, the larger, hydrophobic residue Leu206 position may disrupt Phe381, which helps form the indole-binding hydrophobic pocket. Our exchange of the Leu244 with a Ser244 in MpILR1 L244S altered both substrate recognition and hydrolysis quite dramatically (Table 2). Therefore, we must conclude that this mutation artificially evolved MpILR1 toward what a common progenitor gene would eventually become in vascular plants. We are confident that we have uncovered one clue that helps to bridge the evolutionary gap between the bryophyte and tracheophyte hydrolases. We can demonstrate a clear line of heredity in the homologues that comprise the M20D peptidase family. The charophytic alga K. flaccidum has been characterized as possessing many of the genes that would eventually be required for the evolution of terrestrial plants (Hori et al., 2014). Among these important terrestrial genes were the M20D hydrolases that would eventually help regulate auxins. We have found a single orthologue (GAQ ) for MpILR1 in K. flaccidum (Fig. 1, 2), displaying a 59.5% similarity to the liverwort protein. We can track the source of this enzyme further back in time to cyanobacteria. The cyanobacterium H. byssoidea also carries a homologue (KIF ) for MpILR1 (Fig. 1, 2), with both sharing a 47.6% 16

17 Campanella et al similarity. More interestingly, both the K. flaccidum and H. byssoidea orthologues have the same conserved manganese metal-ion-binding structure as the M20D peptidases found in all plantae. Based on the phylogenetic analysis (Figs. 1, 5) and structural homology, we propose an evolutionary model (Fig. 6) for M20D amidohydrolases. We believe that the source of the M20D peptidase family of enzymes in tracheophytes can be traced directly back to liverwort, and that we can further track the enzyme back to charophytes and cyanobacteria. MpILR1 is further supported as the antecedent to the M20D peptidase family because of its highly conserved intron/exon structure (Fig. 4), which is mirrored in evolution up through angiosperms. It should be noted that MpILR1 lacks the endoplasmic reticulum (ER)-localization signal (KDEL or HDEL) seen in the majority of angiosperm amidohydrolases. Campanella et al. (2014) observed that no characterized gymnosperm M20D peptidase possesses an ER-localization signal. We think that this signal did not evolve until after flowering plants arose. Sanchez et al. (2016) confirmed that in Arabidopsis, the ILR1-like family of hydrolases regulates whole-cell auxin homeostasis from the ER. The two uncharacterized auxin conjugate hydrolases from the early diverging flowering plant Amborella trichopoda (XP_ and XP_ ) contain HDEL signals, so we can trace the evolutionary origin of this sequence to at least the first known eudicot. Since a large proportion of angiosperm M20D hydrolases possess an ERlocalization signal, we speculate that some aspect of flowering plants called for selection of greater homeostatic regulation of auxin Auxin Amidohydrolase Redundancy in Plantae Although more recently evolved tracheophytes have multiple paralogues of the auxin amidohydrolases (Table 1), liverwort contains just the single copy of MpILR1. Since there is 17

18 Campanella et al little evidence that MpILR1 is an active part of auxin regulation in liverwort, this single-copy status probably provides more insight into later evolution of this enzyme family than into liverwort evolution per se. We know that by the time ferns evolved, those first tracheophytes had at least two copies of this gene family (Table 1). The number of isoforms expands as we move into gymnosperms and eventually flowering plants. Auxin conjugate hydrolysis may have become so important in vascular plants as a regulatory activity that evolutionary selection began to favor those plants that possessed additional paralogues as protection against potential mutation and loss. Alternately, paralogous forms may have evolved to carry out differing regulatory functions in various tissues and/or 18

19 Campanella et al developmental stages. This conjecture is supported by the work of Rampey et al. (2004) and Campanella et al. (2003b, 2008, 2014), which demonstrated that paralogues were expressed at different levels in varying tissues of several species. This change in expression suggests that the paralogues perform tissue-specific functions Conclusion It remains unclear whether auxin conjugates are the substrate of MpILR1. The activity of the wild-type liverwort enzyme against these substrates is ineffective, and therefore it seems possible that other substrates may be better candidates. We are quite interested in testing putative candidate substrates of MpILR1 to identify what native function this enzyme may have in M. polymorpha. Performing a knockout for MpILR1 would allow us to ascertain if the auxin regulatory pathway (% free vs. % conjugated) is altered by its loss. Additionally, we want to examine whether the degradation of auxin conjugates really does play a major role in auxin homeostasis in liverwort. Further proteomic studies with MpILR1 can determine what structural changes in this bryophyte hydrolase led to its evolution into the enzyme eventually seen in tracheophytes. In addition, more in-depth physical analyses are required to determine where alterations have appeared in the MpILR1 active site and surrounding amino acid residues. Further study on moss is required to re-visit the process of auxin regulation in the plant. As stated earlier, previous versions of the published moss genome have been contaminated with DNA from soil bacteria (unpublished data). The most recently published P. patens genome should be examined to determine if it has genes encoding auxin conjugate amidohydrolases. It is possible that the speculation of Ludwig-Müller et al. (2009) was correct and moss is a more 19

20 Campanella et al recently evolved dead end for active hydrolysis, but we need to ascertain whether this is indeed the case. Finally, the same functional analyses need to be conducted with fern hydrolases. Since ferns were the earliest tracheophytes, we need to clarify if their hydrolases functionally fall somewhere between the liverwort and the gymnosperm hydrolases that evolved later. In short, are fern hydrolases more like the hydrolases in tracheophytes or bryophytes in function and hydrolysis? Materials and Methods Detection of a Liverwort Hydrolase Homologue A BLAST analysis (Altshul et al., 1990) of the M. polymorpha genome (v3.1) was performed ( The gymnosperm orthologue PsIAR31 DNA sequence (Campanella et al., 2014) was used in this search analysis. One M. polymorpha hydrolase orthologue was identified in the database, MpILR1 (OAE ). A subsequent BLAST search on GenBank with the amino acid sequence of MpILR1 indicated that the newly identified liverwort hydrolase had a greater sequence similarity to ILR-like hydrolases than to the IAR subfamily. All BLAST analyses were performed with default search parameters Cloning and Construction The gene sequences for MpILR1 and MpILR1 L244S were synthesized by Synbio Technologies (Monmouth Junction, New Jersey). MpILR1 L244S was altered at amino acid 244 to code for a serine, whereas the wild-type liverwort sequence coded for a leucine (Supplemental Fig. S1). Both these generated sequences included a T7 inducible IPTG promoter and coded for a 20

21 Campanella et al Shine-Dalgarno sequence in the 5 UTR just upstream of the open reading frames of the hydrolases. These constructed inserts were ligated into the Bam HI site of a puc57 plasmid. We directly transformed these constructs into NovaBlue E. coli cells using heat shock transformation according to the procedure described by Sambrook et al. (1989). Transformants were selected on LB media containing 50 µg/ml ampicillin, using blue-white selection (Sambrook et al., 1989). Plasmids were isolated from transformants by alkaline lysis (Sambrook et al., 1989). Insert orientation and size were determined by endonuclease digestion, electrophoretic analyses using 1% agarose gels, and DNA sequencing of the insert regions using BigDye Terminator Version 3.0 (Applied Biosystems Inc., CA) according to the manufacturer s directions on an ABI model 3730 DNA Analyzer Enzyme Preparation from E. coli MpILR1 and MpILR1 L244S cultures were grown overnight in 5 ml LB medium containing 100 µg/ml ampicillin. From this culture, 2 ml was transferred to a flask containing 50 ml LB medium containing 100 µg/ml ampicillin and 1 mm IPTG for gene induction. Induction was performed for 4 h with continuous shaking of the cultures. Untransformed NovaBlue control cells were grown in the same manner as the induced cells, but without IPTG. Enzyme preparation and enzymatic activity assays were conducted as in our previous studies (Campanella et al., 2003b, 2004, 2008). After collecting the bacterial cells by centrifugation for 10 min at 8000 g, the pellet was re-suspended in 1 ml lysozyme buffer per initial 1 ml bacterial culture (30 mm Tris-HCl, ph 8.0 containing 1 mm EDTA, 20% sucrose and 1 mg/ml lysozyme, Sigma-Aldrich Corp, MO), 1 ml of glycerol, 5 µl of DNase, and 5 µl of RNase. This mixture on ice was sonicated in three cycles using a Microson Ultrasonic Cell 21

22 Campanella et al Disruptor (Misonix Inc., Farmingdale, New York) at full power for 1 min, followed by a 1-min cool down. The extract (100 µl volume per assay) was then directly used for the enzyme assay Hydrolase Enzyme Assays 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 water) of each substrate (final concentration 100 µm, ethanol concentration was always less than 0.1%) (Campanella et al., 2008). 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-Aldrich, Saint Louis, Missouri), and the amide conjugates IBA (Sigma-Aldrich) and IPA (Campanella et al., 2008) with alanine. The assay buffer consisted of 100 mm Tris (ph 8.0), 10 mm MgCl 2, 100 µm MnCl 2, 50 mm KCl, 100 µm PMSF, 1 mm DTT and 10% sucrose (Ludwig-Müller et al., 1996), but for the assays with IAA-Asp as substrate, no DTT was added. The reaction was incubated for 1 h at 40 C and stopped by adding 100 µl 1N HCl, and the aqueous phase was then extracted with 600 µl of ethyl acetate. This was followed by a 1-min centrifugation at 11,700 g. We then transferred the upper organic phase to a new microfuge tube and evaporated it using the SpeedVac (Savant SpeedVac SC110, Savant Refrigerated Condensation Trap RT100, Savant Two Stage VP190, Thermo Fisher Scientific Inc., Waltham, Massachusetts) on a medium setting for 20 min. The evaporated pellet was resuspended in 200 µl of the appropriate running buffer (50% methanol or 50% methanol/1% acetic acid) and analyzed by HPLC according to the procedure described by Campanella et al (2003b). 22

23 Campanella et al The experiments were repeated three to six times using different enzyme preparations. All results represent means of three to six independent experiments + SE. The uninduced cultures were evaluated as controls. The enzymatic activity was calculated as picomoles of IAA, IBA, or IPA released from cultures induced with IPTG minus the values obtained in control nonexpressing cultures Codon Usage Analysis Principle coordinate analysis based on codon usage was performed using the family of auxin conjugate hydrolase orthologues within archaea, eubacteria, and eukaryotes (algae, bryophytes, gymnosperms, and angiosperms). EMBOSS CUSP (Lu et al., 2005) was then employed to obtain the relative abundance values. These values were then input into Vegan (Oksanen et al., 2016) to obtain a dissimilarity matrix (Bray and Curtis, 1957). The data from the dissimilarity matrix was used to perform the principle coordinate analysis via classical multidimensional scaling (R Core Team, 2014; Gower 2015). The two-dimensional plot was created using ggplot2 (Wickham 2009) Global Alignment and Phylogenetic Tree Construction The generation of the similarity matrix by global alignment of the hydrolase orthologues was performed using MatGAT v1.1 (Campanella et al., 2003c). The program was set to default configuration for amino acid analysis. Alignments of all hydrolase homologues were created with the CLUSTAL X v1.81 software, using its default configurations (Thompson et al., 1997). The alignment data obtained from the CLUSTAL X analysis was employed to create phylogenetic trees via the neighbor- 23

24 Campanella et al joining method set to perform 1000 bootstraps (Saitou and Nei, 1987; Felsenstein 1985) and then visualized using TreeView (Page 1996). The outgroup orthologue employed in the analysis was the alga Klebsormidium flaccidum

25 Campanella et al Accession Numbers The NCBI GenBank accession number for the MpILR1 gene is OAE The following genes were employed in the phylogenetic analyses: K. flaccidum (GAQ79540), Arabidopsis (NP_ ), H. byssoidea (KIF ), fern (XP_ , XP_ ), spruce (EF , ED ), pine (TC75408,CT68471), tomato (XP_ ), tobacco (XP_ ), Medicago (XP_ ), potato (XP_ ), barley(baj ), rice (XP_ ), wheat (AAU ), corn (NP_ ) Acknowledgements The authors would like to thank Scott Kight and Jutta Ludwig-Mueller for their advice and encouragement. We would also like to thank Lisa Campanella for her help in editing this manuscript and Rich Skibitski, whose moss studies led to this research. This work was supported by a Margaret and Herman Sokol Fellow Award, #07A Supplemental Data Supplemental Figure 1. Sequence of MpILR1 along with the promoter structure that was inserted into the BamH1 site of the puc57 construct Figure Legends Figure 1. Amino acid sequence similarity matrix of ILR1 orthologues from various species. The matrix was generated with MatGAT v1.1 by using the default values for protein analysis. The bold values are those directly related to MpILR1 similarity to the homologues. 25

26 Campanella et al Figure 2. Principal coordinate analysis of codon usage to determine if MpILR1 was a bacterial contaminant acquired during genomic sequencing. The two-dimensional plot was created using ggplot2 (Wickham 2009). MpILR1 is clearly positioned in the eukaryotic section of the plot Figure 3. Amino acid alignment of several orthologue amidohydrolases. Alignment was performed using CLUSTAL X v1.81. Arrows indicate conserved amino acids (Cys137, His139, Glu173, His197, and His397). The star indicates where the Leucine was substituted in MpILR1 L244S with a serine residue Figure 4. Intron/exon structure of MpILR1. The intron positions for this figure were generated using CLUSTAL X using default settings. The arrow indicates the position of the active site Leu244 in the MpILR1 sequence Figure 5. Neighbor-joining phylogram of the ILR1 protein family members. CLUSTAL X was employed for alignment, and bootstrapping was performed 1000 times. The outgroup used in this phylogenetic analysis is the uncharacterized putative amidohydrolase from Klebsormidium flaccidum. The two important roots are in Bold type Figure 6. Proposed model of the M20D peptidase molecular evolution from cyanobacterium up through vascular plants. Mosses appear with a question mark because, although they seem to have high levels of conjugation (Sztein et al., 1999), it is still unclear whether they possess 26

27 Campanella et al homologous auxin amidohydrolases in this evolutionary path. This model was partly based on that suggested by Cooke et al. (2002). 441 Table 1. Copy-number polymorphisms in the auxin conjugate hydrolase family across plantae Division Species Paralogue hydrolases Monocot (angiosperm) Zea mays 11* Oryza sativa 9* Hordeum vulgare 7* Triticum aestivum 3* Eudicot (angiosperm) Vitis vinifera 15* Glycine max 12* Lycopersicum esculentum 7* Solanum tuberosum 7* Arabidopsis thaliana 7 Medicago truncatula 5 Gymnosperm Picea sitchensis 4 Pinus taeda 4* Picea glauca 2* Bryophyte Physcomitrella patens 3 Marchantia polymorpha 1 Charophyte Klebsormidium flaccidum 1* 442 Cyanobactera Hassallia byssoidea 1* Presence of homologues was determined by BLAST analyses on the GenBank, TIGR, and Phytozome websites. The asterisks indicate hydrolases that have been identified, but not isolated and characterized. 27

28 Campanella et al Table 2. Enzyme/substrate specificity of the MpILR1 and L244-MpILR1 auxin hydrolase Substrate MpILR1 MpILR1 L244S PtIAR31 MtIAR34 AtILL2* IAA-Aspartate IAA-Alanine IAA-Isoleucine IAA-Leucine IAA-Phe IAA-Valine < IAA-Glycine IBA-Alanine N/A IPA-Alanine N/A N/A N/A All hydrolase values are expressed as picomoles of auxin released per minute per milliliter (average from 4 7 replicate experiments) plus or minus the standard error. Untransformed E. coli (NovaBlue) cells were employed as negative hydrolysis controls. Positive hydrolysis is indicated by bold values. The comparative data for Loblolly pine, Medicago, and Arabidopsis from Campanella et al. (2014, 2008), and LeClere et al. (2002), respectively. *Data from LeClere et al (2002) were expressed as nanomoles of auxin released per minute per milligram of protein. 28

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