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Evolutionary history of the GH3 family of acyl adenylasesin rosids
Rachel A. Okrent • Mary C. Wildermuth
Received: 8 October 2010 / Accepted: 10 April 2011
� Springer Science+Business Media B.V. 2011
Abstract GH3 amino acid conjugases have been identi-
fied in many plant and bacterial species. The evolution of
GH3 genes in plant species is explored using the sequenced
rosids Arabidopsis, papaya, poplar, and grape. Analysis of
the sequenced non-rosid eudicots monkey flower and col-
umbine, the monocots maize and rice, as well as spikemoss
and moss is included to provide further insight into the
origin of GH3 clades. Comparison of co-linear genes in
regions surrounding GH3 genes between species helps
reconstruct the evolutionary history of the family. Com-
bining analysis of synteny with phylogenetics, gene
expression and functional data redefines the Group III GH3
genes, of which AtGH3.12/PBS3, a regulator of stress-
induced salicylic acid metabolism and plant defense, is a
member. Contrary to previous reports that restrict PBS3 to
Arabidopsis and its close relatives, PBS3 syntelogs are
identified in poplar, grape, columbine, maize and rice
suggesting descent from a common ancestral chromosome
dating to before the eudicot/monocot split. In addition, the
clade containing PBS3 has undergone a unique expansion
in Arabidopsis, with expression patterns for these genes
consistent with specialized and evolving stress-responsive
functions.
Keywords GH3 � Rosids � Phylogeny � Synteny � Acyl
adenylase � Salicylic acid � Phytohormone
Abbreviations
Compounds
BTH 1,2,3-benzothiodiazole-7-carbothioic acid S-methyl
ester
IAA Indole-3-acetic acid
JA Jasmonic acid
SA Salicylic acid
Genes
bZIP Basic-domain leucine-zipper
ERF Ethylene response factor
GDG1 GH3-like defense gene 1
GH3 Gretchen Hagen 3
ICS1 Isochorismate synthase 1
JAR1 Jasmonic acid resistant 1
PBS3 avrPphB susceptible 3
WIN3 HopW1-1-interacting 3
Organisms
Ac Aquilegia coerulea (columbine)
At Arabidopsis thaliana
Cp Carica papaya (papaya)
Mg Mimulus guttatus (monkey flower)
Os Oryza sativa (rice)
Accession numbers: AtGH3.1, At2g14960; AtGH3.2, At4g37390;
AtGH3.3, At2g23170; AtGH3.4, At1g59500; AtGH3.5, At4g27260;
AtGH3.6, At5g54510; AtGH3.7, At1g23160; AtGH3.8, At5g51470;
AtGH3.9, At2g47750; AtGH3.10, At4g03400; AtGH3.11,
At2g46370; AtGH3.12, At5g13320; AtGH3.13, At5g13350;
AtGH3.14, At5g13360; AtGH3.15, At5g13370; AtGH3.16,
At5g13380; AtGH3.17, At1g28130; AtGH3.18, At1g48670;
AtGH3.19, At1g48660.
Electronic supplementary material The online version of thisarticle (doi:10.1007/s11103-011-9776-y) contains supplementarymaterial, which is available to authorized users.
R. A. Okrent � M. C. Wildermuth (&)
Department of Plant and Microbial Biology, University
of California, 221 Koshland Hall, Berkeley 94720, USA
e-mail: mwildermuth@berkeley.edu
Present Address:R. A. Okrent
Department of Botany and Plant Pathology, Oregon State
University, Corvallis, OR 97331, USA
123
Plant Mol Biol
DOI 10.1007/s11103-011-9776-y
Pp Physcomitrella patens (moss)
Pt Populus trichocarpa (poplar)
Sm Selaginella moellendorffii (spikemoss)
Vv Vitis vinifera (grape)
Zm Zea mays (maize)
Terms
ML Maximum likelihood
MP Maximum parsimony
NJ Neighbor joining
PID Percent identity
WGD Whole genome duplication
Introduction
GH3 (Gretchen Hagen 3) genes were originally identified
in Glycine max (soybean) as responsive to the phytohor-
mone auxin (Hagen et al. 1984) and have since been
identified in many plant species (Terol et al. 2006). Several
Arabidopsis thaliana genes were identified in genetic
screens for altered phytohormone-mediated responses to
auxin [e.g., DFL1 (Nakazawa et al. 2001)], or jasmonic
acid [e.g., JAR1 (Staswick et al. 1992)]. However, the
molecular function of these genes remained unknown until
Staswick et al. identified structural similarity between the
A. thaliana GH3 and the firefly luciferase-like superfamily
of proteins (Staswick et al. 2002). The firefly luciferase-
like superfamily, also called the adenylate-forming super-
family, is a diverse group of enzymes that catalyzes the
addition of AMP to carboxyl groups on a wide variety of
substrates. This family includes nonribosomal peptide
synthetases, 4-coumarate-CoA ligases, acyl-CoA ligases,
and oxidoreductases (Conti et al. 1996). These enzymes
typically contain three conserved motifs that form a bind-
ing pocket for AMP and the substrate (Chang et al. 1997).
Staswick et al. identified the three conserved motifs in the
A. thaliana GH3 proteins. Furthermore, in vitro activity
assays revealed that one, JAR1 (GH3.11), catalyzes the
addition of amino acids to the plant hormone jasmonic acid
(Staswick et al. 2002) and that several others catalyze the
addition of amino acids to auxin (Staswick et al. 2005).
PBS3 (GH3.12), among others, was not active on any of
the phytohormone substrates tested (Staswick et al. 2002).
Our subsequent work determined that 4-substituted ben-
zoates serve as substrates of PBS3 (Okrent et al. 2009).
Previously published phylogenetic trees constructed
using distance methods divided plant GH3 proteins into
three major clades, identified as Groups I, II, and III (Felten
et al. 2009; Staswick et al. 2002; Terol et al. 2006). Groups
I and II contained genes from many more species than did
Group III, which contained genes from only three species,
Arabidopsis thaliana, Brassica napus (rapeseed), and
Gossypium hirsutum (cotton), all in the Eurosids II subc-
lade of the rosids superfamily of eudicotyledonous plants
(Terol et al. 2006). Substrate specificity of assayed proteins
tended to correspond to these phylogenetic relationships
(Staswick et al. 2002, 2005). The JAR1 enzyme active on
JA was placed in GH3 Group I (comprised of 2 GH3 genes
in A. thaliana), with GH3 enzymes active on IAA in Group
II (8 AtGH3 genes), and enzymes including PBS3 active on
neither of these compounds in Group III (9 AtGH3 genes).
We are interested in the evolutionary history of the GH3
family as a means of gaining insight into the evolved
functions of these enzymes in plants. As mentioned above,
this family of enzymes catalyzes the amino acid conjuga-
tion of small molecules, including the hormones IAA and
JA. In doing so, these GH3 proteins alter the activity of the
hormone and its extensive impact on plant metabolism and
physiology. For example, JAR1 catalyzes the conjugation
of Ile to JA forming JA-Ile, the active form of the hormone,
resulting in the degradation of a JA repressor protein and
the subsequent activation of downstream transcriptional
responses (Chini et al. 2007). The function of the Group III
enzymes has remained more elusive, as a substrate for only
one enzyme, PBS3, has been identified (Okrent et al.
2009). Though PBS3 does not act directly on the phyto-
hormone SA, its function is required for full activation of
SA-dependent defense responses (Nobuta et al. 2007;
Jagadeeswaran et al. 2007; Lee et al. 2007). If the Group III
GH3 genes were only present in a small group of related
species, it would suggest the encoded enzymes evolved a
new function. Possibly, this function, e.g., acting on a
unique substrate, would be specifically required by these
species or confer a growth or reproductive benefit.
Herein, we explore the evolutionary history of the GH3
family, focusing particularly on Group III. The recent
sequencing of multiple plant genomes coupled with new
computational tools such as the CoGe suite of comparative
genomics programs (Lyons and Freeling 2008; Lyons et al.
2008) allows us to leverage information from whole gen-
omes to infer descent from a common ancestral gene by
analyzing co-linearity of neighboring genes. We performed
our analysis focusing primarily on genome sequence data
from the rosids Arabidopsis thaliana and Arabidopsis
lyrata, order Brassicales; Carica papaya (papaya), order
Brassicales; Populus trichocarpa (poplar), order Mal-
pighiales; and Vitis vinifera (grape), order Vitales. For
comparison, we also used genome data from the asterid
Mimulus guttatus (monkey flower); order Lamilaes; the
basal eudicot Aquilegia coerulea (columbine), order Ran-
unculales; the monocot grasses Oryza sativa (rice) and Zea
mays (maize); the lycophyte Selaginella moellendorffii
(spikemoss); and the moss Physcomitrella patens (Fig. 1).
This syntenic analysis is coupled with investigation of
expression patterns of the GH3 genes and available
Plant Mol Biol
123
functional information to gain insight on potential gene
function. As detailed below, we find that contrary to past
reports, Group III GH3 enzymes descended from a com-
mon ancestral chromosome dating to before the eudicot/
monocot split and are a sister taxa to the Group II IAA-
conjugating enzymes. Furthermore, our analyses find the
subsequent expansion of Group III GH3s to be consistent
with a role in response to (a)biotic stress. Our identification
of syntelogs (syntenic orthologs) of key Group I, II, and III
members in agronomically-important species allows one to
prioritize those genes for translational research. Moreover,
analysis of conserved genes in syntenic regions may pro-
vide insight on ancient and evolving GH3 syntelog func-
tion(s), as we have explored for PBS3.
Materials and methods
Identification of GH3 genes and syntenic regions
flanking GH3 genes
The CoGe platform for comparison of genome sequences
(http://www.synteny.cnr.berkeley.edu/CoGe/; Lyons and
Freeling 2008) was used to identify regions of potential
synteny between Arabidopis thaliana (TAIR, v9) and
Arabidopsis lyrata (JGI, v1) and other sequenced plant
genomes, including Aquilegia coerulea (JGI, v1), Carica
papaya [v0.4, ASPGB draft genome (Ming et al. 2008)],
Mimulus guttatus (JGI, v1), Oryza sativa [TIGR v5,
(Ouyang et al. 2006)], Physcomitrella patens [JGI v1.1,
(Rensing et al. 2008)], Populus trichocarpa [JGI, v2
(Tuskan et al. 2006)], Selaginella moellendorffii (JGI v1),
Vitis vinifera [v1, French-Italian Public Consortium for
Grapevine Genome Characterization (Jaillon et al. 2007)],
and Zea mays cultivar B73 [Maize sequence.org v2 (Sch-
nable et al. 2009)]. The regions of potential synteny com-
piled in (Lyons et al. 2008) were used as a starting point,
modified with new genome sequence data, evaluated for
accuracy, and expanded with additional analyses. The
V. vinifera genome, less subject to rearrangements, dupli-
cations and gene loss than other genomes (Jaillon et al.
2007; Semon and Wolfe 2007), was used as a bridge
between A. thaliana and the other genomes to identify
regions of co-linearity and possible synteny. It should be
noted that genes from some of the sequenced plants have
not yet been assigned to chromosomes (Phytozome 6.0).
Four Arabidopsis GH3 genes were used as seeds to
identify GH3 genes in the other eudicot genomes. The CDS
sequences of PBS3 (AtGH3.12, At5g13320), DFL1
(AtGH3.6, At5g54510), JAR1 (AtGH3.11, At2g46370), and
DFL2 (AtGH3.10; At4g03400) were used as BLAST seeds
against papaya, grape, poplar, monkey flower, and colum-
bine in CoGe Blast. Similarly, Arabidopsis and rice GH3
sequences were used to identify homologs in maize, and
moss GH3 sequences were used to identify homologs in
spikemoss. Additional BLASTN searches were performed
with the CDS sequence from each species against the gen-
ome of origin to find any other possible matches, and results
were checked against genes containing the keyword
‘‘GH3’’ in Phytozome v6 (http://www.phytozome.net/) from
Department of Energy’s Joint Genome Institute and the
Center for Integrative Genomics. Sequence length and gene
models were also retrieved from the Phytozome website and
compared to BLASTX results. The sequences were aligned
using MUSCLE (Edgar 2004), visualized in JalView and
evaluated for global alignment and presence of AMP-bind-
ing motifs (Chang et al. 1997).
As two divergent haplotypes of Selaginella were
sequenced, a non-redundant set of sequences was com-
posed using a gene set from JGI (http://www.genome.jgi.-
psf.org/Selmo1.info.html) and by removing nearly identi-
cal sequences. Several sequences were found to be
incomplete. GSVIV00026990001 (VvGH3.7) was missing
motif II and the protein sequence was shorter than expected
by approximately 200 amino acids based on comparison
with other GH3 proteins, typically around 600 amino acids
long. BLAST searches found peptides and ESTs containing
the missing sequence information (ESTs gi 110368758 and
gi 110698541 and peptide gi 225454466). The corrected
sequence length and exon number are shown in Table 1.
Two papaya sequences, EVM prediction supercon-
tig_1065.2 (CpGH3.3) and EVM prediction supercon-
tig_9.204 (CpGH3.4) were both truncated due to missing
Paleohexaploidy
Paleotetraploidy
Vitis vinifera
Mimulus guttatus
Aquilegia coerulea
Zea mays
Oryza sativa
Selaginella moellendorffii
Physcomitrella patens
Populus trichocarpa
Arabidopsis thaliana
Carica papaya
10
19
8
13
6
13
2
13
20
6
9
3
rosids
eudicots
monocots
Fig. 1 Plant phylogeny showing number of GH3 genes in modern
species and location of paleohexaploidy and paleotetraploidy events
in lineages of interest. Figure adapted from Freeling (2009) with
phylogenetic information from the Missouri Botanical Garden’s
Angiosperm Phylogeny Project and Phytozome v6
Plant Mol Biol
123
sequence data. Some additional base pairs in the C-termi-
nus of the p1065.2 gDNA sequence were identified as
potentially coding, and added to the CDS sequence.
However, this correction did not account for all of the
missing sequence at the C-terminus. No ESTs were iden-
tified that contain the missing regions for either of the
papaya sequences, so they remain incomplete.
Sequence alignment and phylogenetic tree construction
CDS and peptide sequences were aligned with MUSCLE
(Edgar 2004) via the online phylogeny.fr platform (http://
www.phylogeny.fr; Dereeper et al. 2008) using default
parameters. Distance (BioNJ), maximum likelihood
(PhyML) and maximum parsimony (TNT) tree construc-
tion methods were used and MEGA 4.0 (Tamura et al.
2007) was employed to visualize and annotate the phylo-
genetic trees. Sequences missing AMP binding motifs or
the highly conserved C-terminus were omitted from
alignments and phylogenetic tree construction. DNA
regions containing sequences related phylogenetically were
tested for synteny using the GEvo tool of the CoGe
browser, described above.
Analysis of expression data
The expression patterns of AtGH3 genes were explored
using tools from Genevestigator (Zimmermann et al.
Table 1 GH3 genes in the rosids Populus trichocarpa, Vitis vinifera, and Carica papaya and their relationship to Arabidopsis thaliana genes
through common position on an ancestral chromosome
Namea Locusb Pos.c Protein Lengthb Exonsb Groupd At syntelog (s)d
PtGH3.1 POPTR_0007s10350 7 597 3 II AtGH3.2, AtGH3.3
PtGH3.2 POPTR_0009S09590 9 690 3 II AtGH3.1
PtGH3.3 POPTR_0001S30560 1 596 3 II AtGH3.1
PtGH3.4 POPTR_0013S14740 13 608 3 II –
PtGH3.5 POPTR_0011S13330 11 611 3 II AtGH3.5, AtGH3.6
PtGH3.6 POPTR_0001S43990 1 611 3 II AtGH3.5, AtGH3.6
PtGH3.7 POPTR_0001S12850 1 606 4 III AtGH3.12, AtGH3.17
PtGH3.8 POPTR_0003S15970 3 594 5 III AtGH3.12, AtGH3.17
PtGH3.9 POPTR_0002S20790 2 596 4 III AtGH3.9
PtGH3.10 POPTR_0013S14050 13 595 4 I AtGH3.10
PtGH3.11 POPTR_0019S13450 19 595 4 I AtGH3.10
PtGH3.14 POPTR_0014S09120 14 576 4 I AtGH3.11
PtGH3.15 POPTR_0002S16960 2 576 4 I AtGH3.11
VvGH3.1 GSVIV00007718001 3 598 4 II AtGH3.1
VvGH3.2 GSVIV00019610001 7 600 3 II AtGH3.2, AtGH3.3
VvGH3.3 GSVIV00027472001 19 605 4 II AtGH3.5 AtGH3.6
VvGH3.4 GSVIV00026120001 12 588 5 II –
VvGH3.5 GSVIV00027964001 7 596 4 III AtGH3.9
VvGH3.6 GSVIV00026000001 12 592 4 I AtGH3.10
VvGH3.7 GSVIV00026990001e 15 583 4 I AtGH3.11
VvGH3.8 GSVIV00006220001 1 593 5 III AtGH3.12, AtGH3.17
CpGH3.1 EVM supercontig_292.1 292 599 3 II AtGH3.1
CpGH3.2 EVM supercontig_6.74 6 599 3 II AtGH3.2 AtGH3.3
CpGH3.3 EVM supercontig_1065.2 1065 455f 3 II AtGH3.5 AtGH3.6
CpGH3.4 EVM supercontig_9.204 9 491f 3 III AtGH3.9
CpGH3.5 EVM supercontig_34.122 34 607 4 I AtGH3.10
CpGH3.6 EVM supercontig_1483.1 1483 591 4 I AtGH3.11
Syntelogs of PBS3 (AtGH3.12) are in bolda Pt names from Felten et. al (2009), PtGH3.15, Vv, Cp and Mg names first described hereb Locus names, protein length and exon number from Phytozome v5.0c Position is the chromosome for Pt and Vv, and supercontig for Cp and Mgd Group and At syntelog from synteny analysis described in Materials and Methodse Gene model in Phytozome v5.0 corrected based on global alignment and EST data, see Materials and Methodsf Sequencing error results in missing sequence
Plant Mol Biol
123
2004), NascArrays (Craigon et al. 2004), the eFP brower
(Toufighi et al. 2005; Winter et al. 2007) (http://www.bar.
utoronto.ca/efp/cgi-bin/efpWeb.cgi) and analysis of the
relevant literature. Experiments in which GH3 genes were
expressed in response to abiotic or biotic stress or hormone
treatments were identified in Genevestigator and the eFP
browser, and data downloaded. Most experiments analyzed
were from the AtGenExpress series (Goda et al. 2008). The
NascArrays experiment reference numbers are shown in
the corresponding data tables in the ‘‘Results’’ section.
Values greater than 2-fold increase relative to control
experiments were reported and significance tested using
student’s t tests at a = 0.05 for experiments with three
replicates. Experiments with two replicates were manually
examined for reproducibility of experimental and control
samples. PtGH3 and VvGH3 expression patterns were
analyzed using the Plant Expression Database [Plexdb,
http://www.plexdb.org (Wise et al. 2007)].
Analysis of transcription factor binding motifs
Potential transcription factor binding sites 1 kb upstream of
AtGH3 transcriptional start sites were identified using the
Arabidopsis cis-regulatory element database (AtcisDB,
http://www.arabidopsis.med.ohio-state.edu/AtcisDB/;
Molina and Grotewold 2005) of the Arabidopsis Gene
Regulatory Information Server from Ohio State University.
Several of the tandemly duplicated genes have fewer than
1 kB between the next gene: AtGH3.13 (100 bp),
AtGH3.15 (288 bp), AtGH3.16 (711 bp) and AtGH3.18
(989 bp). Promoter motifs associated with transcription
factor families of interest were tallied and summarized.
These plant transcription factors include WRKYs, basic-
domain leucine-zippers (bZIPs), MYBs, and dehydration
response element binding proteins (DREBs) known to
mediate plant response to biotic stress (Singh et al. 2002).
Furthermore, ethylene responsive element binding factors
(ERFs), auxin response factors (ARFs) and MYC2 tran-
scription factors (Dombrecht et al. 2007) were included to
reflect responses mediated by the hormones ethylene,
auxin, and jasmonate, respectively. Cis-acting regulatory
elements are defined as follows: WRKY transcription
factors recognize the W-box: ttgact/c; bZIPs recognize
actcat (ATB2/AtbZip53) and acacttg (DPBF1&2) motifs;
the MYB and MYB-like transcription factors bind MYB
(aaccaaac, taactaac) and aaatct (MYB-related CCA1)
motifs; DREBs recognize tgccgacaa, gaccgacct, and aacc-
gacca motifs; ERFs bind the GCC-box (gccgcc), ARF1
binds tgtctc; and MYC2 binds the G-box (cacgtg), T/G-box
(cacgtt), and cacatg.
Results
Identification of GH3 genes
Potential orthologs of the AtGH3 genes were identified by
BLASTN search using CoGe Blast with AtGH3, OsGH3,
and PpGH3 genes as seeds, aligned using MUSCLE (Edgar
2004), visualized in JalView and evaluated for global
alignment and presence of AMP-binding motifs and the
highly conserved C-terminal domain (see Materials and
Methods). For the rosids, in addition to the 19 Arabidopsis
thaliana GH3 genes, 6 GH3 genes were identified in Carica
papaya, 13 in Populus trichocarpa [one newly described
gene plus 12 described in (Felten et al. 2009)], and 8 GH3
genes were identified in Vitis vinifera (Fig. 1, Table 1).
For comparison, we also identified GH3 genes in non-
rosid eudicots Mimulus guttatus (6 GH3 genes) and Aqui-
legia coerulea (10 genes), as well as in the monocot grasses
Zea mays (13 GH3 genes) and Oryza sativa [13 genes;
previously described in (Jain et al. 2006)], and in Selagi-
nella moellendorffii (20 GH3 genes) and Physcomitrella
patens [2 genes; previously described in (Bierfreund et al.
2004) and (Ludwig-Muller et al. 2009)] (Fig. 1, Online
Resource 1). It should be noted that there may be additional
M. guttatus and C. papaya GH3 genes, as the sequencing
and annotation of those genomes are still incomplete.
Loss and gain of GH3 genes in rosids
Analysis of synteny is complicated by gene duplication,
either due to whole genome duplication (WGD) or local
duplication, gene loss or insertion (Lyons et al. 2008). Plant
genomes are heavily duplicated, with WGD events quite
common in the evolutionary history of many plant lineages.
Following episodes of genome duplication, selective gene
loss, called fractionation, is typically observed (Freeling
2009). As shown in Fig. 1, the rosids all show evidence of
a pre-rosid hexaploidy event with subsequent fractionation.
Though the exact classification of V. vinifera remains
uncertain, sequence comparisons of chloroplast genomes
suggest that V. vinifera is a basal rosid (Jaillon et al. 2007;
Jansen et al. 2006) as depicted. Although A. thaliana has
the smallest genome of any sequenced plant, two rounds of
WGD have occurred since its divergence from the primary
rosid lineage (Blanc et al. 2003). One round of WGD has
also occurred in the P. trichocarpa lineage. This can lead to
as many as four copies of A. thaliana and two of
P. trichocarpa for each V. vinifera or C. papaya gene.
Though Fig. 1 does not show a WGD event as part of
M. guttatus lineage, several members of the Mimulus genus
have been shown to have evidence of polyploidy (Wu et al.
Plant Mol Biol
123
2007). Completion of the M. guttatus annotation will allow
future resolution of this issue.
As noted on Fig. 1, our analysis indicates that the
common ancestor prior to the pre-rosid hexaploidy WGD
event had 3 GH3 genes, leading to nine after the hexa-
ploidy WGD event, one of which was lost in the lineage
prior to the divergence of grape. V. vinifera has retained all
8 GH3 genes, but the other rosids analyzed have lost
multiple genes (Table 2). Uniquely, A. thaliana lost 21
genes but also gained 7 GH3 genes, due to whole genome
duplication, fractionation, local duplication and transposi-
tion. Of particular interest, the genes gained are all in
Group IIIA defined below.
Identification of GH3 syntenic sets
Using the GEvo tool of the CoGe genome comparison
browser (Lyons and Freeling 2008), eight GH3 syntenic
sets were identified, incorporating ten genomic regions of
Arabidopsis thaliana that flank GH3 genes. These include
several areas of A. thaliana chromosomes 1, 2, 4, and 5 and
account for 14 of the 19 A. thaliana GH3 (AtGH3) genes.
The gene names, locus, chromosome, contig, or scaffold
number, predicted protein length, number of exons, and
Arabidopsis syntelog are shown in Table 1 for the rosids
and Online Resource 1 for the others.
As an example, Fig. 2 shows part of the Arabidopsis
PBS3 (AtGH3.12/At5g13320) syntenic set including
approximately 40 kB of Arabidopsis chromosome 5 with
100 kB of V. vinifera chromosome 1, 90 kB P. trichocarpa
chromosome 1, and 60 kB of P. trichocarpa chromosome
3. The Arabidopsis chromosome 5 region contains PBS3
(AtGH3.12, At5g13320) and four other AtGH3 (AtGH3.13-
16) genes. Though not shown in Fig. 2, 70 kB of Arabid-
opsis chromosome 1 containing AtGH3.17 is also syntenic
(see Table 3 and Online Resource 2). Of the rosids, only
C. papaya did not share a region of synteny with PBS3;
however, this needs to be reexamined once the final com-
pleted and annotated genome is available. Regions of
M. guttatus showed evidence of synteny with the region
surrounding PBS3 (see Table 3), however, none of these
M. guttatus regions contain a GH3 sequence. It remains
possible, however, that a GH3 sequence resides in the
missing region of M. guttatus scaffold 39 (open boxes in
Table 3). As shown in Online Resource 2, Aquilegia
coerulea does contain a syntenic region (mapped to scaf-
fold 49) and GH3 gene, AcGH3.6. Similarly, a region of
Oryza sativa chromosome 11 containing OsGH3.13
(Os11g32520) (Terol et al. 2006) and a region of Zea mays
containing ZmGH3.13 demonstrate co-linearity of genes
with the region containing Arabidopsis PBS3 (Online
Resource 2). No syntenic region is detectable between
S. moellendorffii or P. patens.
A detailed comparison of the syntenic regions flanking
PBS3 shows that the five AtGH3 genes in the Arabidopsis
chromosome 5 region correspond to a single gene in each
of the other genomes. This suggests that these additional
Arabidopsis GH3 genes result from local duplication. Two
additional genes (At5g13330 and At5g13340, annotated as
an ERF/AP2 transcription factor family member and of
unknown function, respectively) are located between PBS3
and the other four AtGH3 genes. One of these, the gene
encoding an ERF/AP2 transcription factor, also has syn-
telogs in many of the species examined. In total, in addition
to the GH3 genes, seven Arabidopsis genes in the chro-
mosome 5 region and three in the chromosome 1 region
display synteny to genetic regions of other species. These
genes and their annotations are summarized in a slightly
abbreviated version for the eudicots in Table 3 and more
fully for all species examined in Online Resource 2.
Five A. thaliana, one P. trichocarpa (PtGH3.4), one
A. coerulea (AcGH3.1) and two O. sativa GH3 genes
(OsGH3.7 and OsGH3.12) are not members of syntenic sets
(Online Resources 1 and 3); there is no detectable co-line-
arity between surrounding genes and GH3 genes from other
species. In addition, it is not possible to detect co-linearity
between chromosomal regions surrounding the S. mo-
ellendorfii and P. patens genes and the other species studied
(data not shown). Four of the five Arabidopsis genes not in a
syntenic set (AtGH3.7, AtGH3.8, AtGH3.18, AtGH3.19) are
in Group IIIA (defined below), which contains PBS3, and
can be explained by local duplication and gene insertion
(Fig. 3). AtGH3 genes, AtGH3.18 (At1g48670) and
AtGH3.19 (At1g48660), as well as a severely truncated GH3
gene (At1g48690), likely arose from insertion and duplica-
tion (Fig. 3) and there are several retrotransposons nearby
(e.g. At1g48680). AtGH3.7 (At1g23160), most similar in
protein sequence to PBS3, is present in A. lyrata but not
papaya, suggesting that it was inserted into an ancestor of
Table 2 Loss and gain of GH3 genes in each syntenic group since
divergence from common ancestor
Syntenic group At Cp Pt Vv
IA -3 NC -1 NC
IB -3 NC NC NC
IIA1 -2 NC -1 NC
IIA2 -2 NC NC NC
IIB1 -2 NC NC NC
IIB2 -4 -1 -1 NC
IIIA 22/17 21 21 NC
IIIB -3 -1 NC NC
Total 19 5 12 8
Group in bold contains PBS3. NC no change
Plant Mol Biol
123
A. thaliana before the divergence from A. lyrata but after
papaya (data not shown). The region surrounding AtGH3.8
(At5g51470) contains many stress response genes, including
PBS2, also known as RAR1, which was identified in the same
mutant screen for altered disease resistance as PBS3 (War-
ren et al. 1999) (data not shown, can be recapitulated
http://www.genomevolution.org/r/37d). Regions of V. vinif-
era chromosome 16 and P. trichocarpa chromosomes 12
and 15 display co-linear genes with the region around
AtGH3.8 (At5g51470), although no GH3 gene is present,
suggesting that AtGH3.8 was inserted at this site. Though
AtGH3.4 (At1g59500) is grouped with the IIA GH3 proteins
(below), synteny was ambiguous as only one other gene in
the surrounding region matched genes from other species.
Classification of GH3 proteins into groups based
on synteny and phylogenetic relationships
Corrected GH3 protein sequences were aligned using
MUSCLE (Edgar 2004), and curated by removing gaps
manually or using Gblocks (Castresana 2000) the phylog-
eny.fr server (Dereeper et al. 2008) as described in
‘‘Materials and methods’’. Alignments were constructed
and clustered into phylogenetic trees using maximum
parsimony (TNT), maximum likelihood (PhyML), and
neighbor joining (BioNJ) algorithms. The tree found to best
correspond to the syntenic relationships identified above
was constructed using a multiple sequence alignment
curated using Gblocks with relaxed settings and PhyML.
This phylogenetic tree comprised of the eudicot GH3
sequences is shown in Fig. 4, with the complete phyloge-
netic tree including eudicot, monocot, moss and spikemoss
sequences provided as Online Resource 3.
The eight sets of GH3 syntenic genes are distributed
between Groups I, II, and III. The proteins from all species
other than S. moellendorffii and P. patens separate into
these three Groups in the phylogenetic trees, with each
syntenic set containing one V. vinifera protein. Each of the
two Group I sets contains one VvGH3, AtGH3 (AtGH3.11/
JAR1 in Set IA and AtGH3.10/DFL2 in Set IB), CpGH3,
MgGH3, and AcGH3 protein and two poplar proteins, with
Set IA also including monocot grass syntelogs.
Group II contains four VvGH3 proteins, each of which
corresponds to a syntenic subset of sequences (IIA1, IIA2,
IIB1, and IIB2). Within IIB, IIB1 contains eight proteins
including AtGH3.5/WES1 and AtGH3.6/DFL1, other rosid
proteins, as well as proteins from Mimulus and Aguilegia,
while IIB2 is comprised only of VvGH3.4 and PtGH3.4.
Within subgroup IIA, the sequences are more evenly dis-
tributed between IIA1 and IIA2 syntentic sets, with
Pt 2
At
Vv
Pt 1
AtGH3.16AtGH3.14AtGH3.13 AtGH3.15PBS3
AtGH3.12
Fig. 2 Synteny between the region of Arabidopsis chromosome 5
surrounding PBS3 and grape and poplar chromosomes. A screenshot
of the BLASTz output from the CoGe browser is shown. Each large
horizontal bar represents one genomic region, with the dashed linedividing the top (50 on left) and bottom (50 on right) strand. The
genome origin is indicated on the right (At Arabidopsis thaliana, Vv
Vitis vinifera, Pt Populus trichocarpa). The colored arrows represent
gene models: green are CDS, blue are RNA, and gray are introns. The
areas of similarity from BLASTz between genomic regions are shown
as colored blocks above or below the gene models. Each pairwise
comparison is shown in a different color. Brown and pink lines are
drawn between similar regions of At and the other species. Linesconnecting other pairwise comparisons were omitted for clarity. The
analysis can be regenerated at http://www.genomevolution.org/r/1d9i.
The 70 kB syntenic region of Arabidopsis chromosome 1 containing
AtGH3.17 is not shown here but is included in Table 3
Plant Mol Biol
123
Ta
ble
3C
om
par
iso
no
fsy
nte
nic
reg
ion
sin
clu
din
gP
BS
3(A
tGH
3.1
2,
At5
g1
33
20
)
At
locu
s1
Ch
5
At
locu
s2
Ch
1
Vv
locu
s1
Ch
1
Pt
locu
s1
Ch
1
Pt
locu
s2
Ch
3
Mg
locu
s1
scaf
fold
39
Mg
locu
s2
scaf
fold
7
Mg
locu
s3
scaf
fold
54
An
no
tati
on
At5
g1
32
50
At1
g2
80
80
No
ne
No
ne
No
ne
No
ne
No
ne
Un
kn
ow
n,
pla
stid
At5
G1
33
20
(AtG
H3
.12
)A
t1g
28
13
0(A
tGH
3.1
7)
GS
VIV
00
00
62
20
00
1(V
vG
H3
.8)
PO
PT
R_
00
01
s12
85
0(P
tGH
3.7
)P
OP
TR
_0
00
3s1
59
70
(PtG
H3
.8)
No
ne
No
ne
PB
S3
,G
H3
fam
ily
pro
tein
At5
g1
33
30
At1
g2
81
60
GS
VIV
00
00
62
01
00
1P
OP
TR
_0
00
1s1
28
20
PO
PT
R_
00
03
s15
94
0N
on
eN
on
eE
RF
/AP
2tr
ansc
rip
tio
nfa
cto
r
At5
g1
33
40
No
ne
No
ne
No
ne
No
ne
No
ne
No
ne
Un
kn
ow
n
At5
g1
33
50
No
ne
No
ne
No
ne
No
ne
No
ne
No
ne
GH
3fa
mil
yp
rote
in
At5
g1
33
60
No
ne
No
ne
No
ne
No
ne
No
ne
No
ne
GH
3fa
mil
yp
rote
in
At5
g1
33
70
No
ne
No
ne
No
ne
No
ne
No
ne
No
ne
GH
3fa
mil
yp
rote
in
At5
g1
33
80
No
ne
No
ne
No
ne
No
ne
No
ne
No
ne
GH
3fa
mil
yp
rote
in
At5
g1
33
90
No
ne
GS
VIV
00
00
62
19
00
1P
OP
TR
_0
00
1s1
28
60
PO
PT
R_
00
03
s15
98
0m
gf0
12
81
5m
No
ne
No
ne
NE
F1
,ch
loro
pla
st
At5
g1
34
00
No
ne
GS
VIV
00
00
62
14
00
1P
OP
TR
_0
00
1s1
28
90
No
ne
mg
f01
61
73
mN
on
eN
on
eP
roto
n-d
epen
den
t
oli
go
pep
tid
etr
ansp
ort
,
pla
stid
No
ne
No
ne
GS
VIV
00
00
62
11
00
1P
OP
TR
_0
00
1s1
29
10
PO
PT
R_
00
03
s16
01
0m
gf0
87
73
mN
on
eN
on
eC
yto
chro
me
P4
50
At5
g1
34
10
No
ne
GS
VIV
00
00
62
10
00
1P
OP
TR
_0
00
1s1
29
20
No
ne
mg
f01
57
87
mN
on
eN
on
eP
epti
dy
l-p
roly
lci
s–tr
ans
iso
mer
ase,
chlo
rop
last
At5
g1
34
20
No
ne
GS
VIV
00
00
62
09
00
1P
OP
TR
_0
00
1s1
29
30
PO
PT
R_
00
03
s16
03
0m
gf0
12
10
0m
No
ne
No
ne
Tra
nsa
ldo
lase
,ch
loro
pla
st
No
ne
No
ne
GS
VIV
00
00
62
08
00
1N
on
eN
on
eN
on
em
gf0
04
55
6m
mg
f01
49
39
mU
nk
no
wn
No
ne
No
ne
GS
VIV
00
00
62
06
00
1N
on
eN
on
eN
on
em
gf0
15
94
6m
mg
f01
43
06
m,
mg
f00
06
51
m,
mg
f00
80
51
m
Un
kn
ow
n
At5
g1
34
30
,
At5
g1
34
40
No
ne
GS
VIV
00
00
62
05
00
1P
OP
TR
_0
00
1s1
29
60
PO
PT
R_
00
03
s16
06
0m
gf0
04
95
9m
No
ne
No
ne
Ub
iqu
ino
l-cy
toch
rom
eC
red
uct
ase
mit
och
on
dri
on
No
ne
At1
G2
81
40
GS
VIV
00
00
62
02
00
1N
on
eN
on
em
gf0
03
85
0m
No
ne
No
ne
Un
kn
ow
n,
chlo
rop
last
Plant Mol Biol
123
AtGH3.1 and AtGH3.3 in IIA1 and AtGH3.2/YDK1 and
AtGH3.3 in IIA1 and AtGH3.1 in IIA2. As the evolu-
tionary distance of the species increases, it is increasingly
difficult to distinguish subsets of IIA and IIB. Though we
did assign subsets of IIA and IIB for Mimulus and
Aguilegia, there is less certainty about this assignment than
for the rosids. For the monocots, we could not parse out
these subsets and differentiate only IIA from IIB (Online
Resource 3). If MgGH3.3 is misassigned and should be
placed in IIA1 instead of IIA2, then a duplication of the
Group II sequences after the divergence of the rosids would
explain the presence of non-rosid species in only one
subset of IIA (i.e. IIA1) and IIB (i.e. IIB1).
Group III contains two VvGH3 proteins, corresponding
to two syntenic sets, both of which contain proteins from
species other than rosids. Set IIIA, contains the Arabidopsis
Syntenic set:IIIA
IIIB
insertion
insertion
local duplication
local duplication
(At2g47750)
(At1g28130)(At5g13360)(At5g13370)
(At5g13380)(At5g13350)(At1g48670)
(At1g48660)(At5g51470)
(At1g23160)
Fig. 3 Maximum likelihood phylogenetic tree of Group III rosid
GH3 proteins, with evidence for expansion in Arabidopsis Group IIIA
detailed. The tree was constructed using PhyML on multiple sequence
alignments from MUSCLE curated using Gblocks with reliability of
internal branch length tested using the aLRT method. Branches with
the same symbol represent a set with evidence of synteny. Sequences
are from the rosid species At Arabidopsis thaliana, Cp Caricapapaya, Pt Populus trichocarpa, and Vv Vitis vinifera. PBS3 is
highlighted
B
II
III
Syntenic sets:IA IB IIA1
IIA2 IIB1
IIB2 IIIA
IIIB
A
B
A
B
A
I
Fig. 4 Maximum likelihood
phylogenetic tree of eudicot
GH3 proteins. The tree was
constructed using PhyML on
multiple sequence alignments
from MUSCLE curated using
Gblocks with reliability of
internal branch length tested
using the aLRT method.
Branches with the same symbol
represent a set with evidence of
synteny. Sequences are from the
species Aq Aquilegia coerulea,
At Arabidopsis thaliana,
Cp Carica papaya, Mg Mimulusguttatus, Pt Populustrichocarpa, and Vv Vitisvinifera. PBS3 is highlighted
Plant Mol Biol
123
proteins PBS3 (AtGH3.12) and AtGH3.13-16 on chromo-
some 5 and AtGH3.17 on chromosome 1. PBS3 syntelogs
in other species include VvGH3.8, PtGH3.7 and PtGH3.8,
AcGH3.6, OsGH3.13 and ZmGH3.13 (Online Resources 2
and 3). Set IIIB, contains AtGH3.9 and a variety of other
eudicot proteins. AtGH3.17 of Set IIIA and AtGH3.9 of Set
IIIB had previously been classified with Group II proteins
(Staswick et al. 2005).
Analysis of GH3 functional data
Examination of expression data and transcription factor
binding motifs in gene promoters complements biochemi-
cal and phenotypic data and can help provide insight into
gene function. For example, the first GH3 genes were
identified as inducible by auxin (Hagen et al. 1984), years
before their function was known. Subsequent analysis
revealed GH3 enzymes that catalyze the conjugation of
amino acids to the auxin IAA regulating IAA activity and
function (Staswick et al. 2005). Most of the available
functional data is for the Arabidopsis GH3 genes/proteins,
the focus of our functional analysis.
Arabidopsis GH3s induced by IAA are in Group II
(Fig. 5). Though all Group II AtGH3 genes are induced by
IAA, only two of the six analyzed contain a cis-acting
regulatory element bound by auxin responsive transcription
factors (ARFs). However, on a percentage basis Group II
promoters are more likely to contain at least one cis-acting
regulatory element bound by an ARF at 33%, compared
with 0 and 20% for Groups I and III, respectively (Online
Resource 4). It should be noted that AtGH3.17 and
AtGH3.9, which we reclassify as Group III above, are not
induced by IAA (Fig. 5.) We find the Group II GH3s are
also induced by (a)biotic stress including pathogens
(Fig. 6) with the exception of the lowly expressed
AtGH3.1. In addition to Group II promoters being enriched
in cis-acting regulatory elements bound by ARFs, they are
also enriched for regulatory elements bound by ethylene
responsive binding factors (ERFs) known to regulate
response to (a)biotic stress (Gutterson and Reuber 2004).
Group II Arabidopsis GH3 proteins are also active on IAA,
with mutants in these genes resulting in an IAA phenotype
when tested (Fig. 5). Of note, AtGH3.5/WES1 is active on
both IAA and salicylic acid (2-hydroxybenzoate) (Staswick
et al. 2002; Park et al. 2007b; Zhang et al. 2007).
In contrast to the Arabidopsis Group II genes, which are
all induced by IAA and encode proteins active on IAA, the
two Group I genes AtGH3.11/JAR1 and AtGH3.10/DFL2
have minimal unifying functional data. Surprisingly,
though JAR1 is active on JA (Staswick and Tiryaki 2004),
JAR1 is not induced by JA, other hormones, or in response
to a diverse set of pathogens (Figs. 5 and 6). jar1 mutants
do however exhibit JA-associated phenotypes and are more
susceptible to pathogens such as Botrytis cinerea that
activate ET/JA-dependent defenses (Ferrari et al. 2003).
AtGH3.10/DFL2 is in a distinct syntenic set from JAR1
and does not appear to function in JA signaling and
response (Fig. 5). Instead, it mediates red light-specific
hypocotyl elongation with its expression controlled by light
(Takase et al. 2003). Interestingly, jar1 mutants also
exhibit far red light insensitivity (Hsieh et al. 2000). Nei-
ther Group I Arabidopsis gene contains cis-acting regula-
tory elements bound by ARFs, ERFs, or dehydration
response element binding proteins (DREBs) in their 1 kb
promoters (Online Resource 4). However, they do contain
other biotic-stress associated cis-acting regulatory elements
including those bound by MYC2 transcription factors. Of
particular interest, the promoter of JAR1 contains 4 MYC2
binding sites. Proteolysis of the JAZ family of JA repres-
sors is mediated by the JA-Ile conjugate whose formation
is catalyzed by JAR1. The proteolysis of the JAZ repressor
then allows for downstream JA-associated gene expression
through MYC2 transcription factors (Dombrecht et al.
2007).
An analysis of publicly available expression data
showed the Arabidopsis Group III GH3 genes tend to have
higher expression then those in Group I and II, excluding
AtGH3.13 and 16, whose expression has not been observed
(Figs. 5 and 6; Online Resource 5). Two of the seven tested
and expressed Group III A. thaliana GH3 genes are
induced by phytohormone treatment (Fig. 5) with five of
the seven induced by pathogen or abiotic stress (Fig. 6).
Group III GH3s tend to contain cis-acting regulatory ele-
ments associated with (a)biotic stress response in their
promoters (Online Resource 4). It is interesting that motifs
bound by DREBs are only present in the promoters of the
Group III genes AtGH3.12/PBS3, AtGH3.7 (the most
similar A. thaliana protein to PBS3), and AtGH3.15. In
addition, PBS3 and AtGH3.7 are induced by osmotic stress
(Fig. 6); AtGH3.15 does not have a gene-specific probe on
the ATH1 array. In terms of enzymatic activity, AtGH3.9
and 17, now classified into distinct syntenic Group III sets
are active on IAA, though their expression is not induced
by it [Fig. 5 (Khan and Stone 2007; Staswick et al. 2005)].
AtGH3.12 (PBS3), a syntelog of AtGH3.17, is not active
on IAA, but on 4-substituted benzoates (Okrent et al.
2009). Its expression is induced by SA [Fig. 5 and (Jaga-
deeswaran et al. 2007)], with mutants exhibiting compro-
mised SA accumulation and pathogen resistance (Okrent
et al. 2009; Lee et al. 2007; Jagadeeswaran et al. 2007).
Substrates for other Arabidopsis Group III members remain
unknown.
In addition to the functional data discussed above for
Arabidopsis GH3 genes, data for P. trichocarpa, V. vinif-
era, and O. sativa provide further evidence for selected
biotic stress induction of Group II and III GH3 members.
Plant Mol Biol
123
For example, in poplar (Populus tremula 9 Populus alba)
tree roots colonized by the ectomycorrhizal fungus (EMF)
Laccaria bicolor, the Group III PBS3 syntelogs PtGH3.7,
and Pt.GH3.8 were induced (Felten et al. 2009). In con-
trast, PtGH3.7 (probeset PtpAffx.140928.1.A1_at) and
PtGH3.8 (probeset PtpAffx.210014.1.S1_at) were not
significantly elevated in response to infection with the
Melampsora rust fungus (Plant Expression Database). No
probesets were identified for the grape Group III members
the PBS3 syntelog VvGH3.8 or the AtGH3.9 syntelog
VvGH3.5; however, the PBS3 syntelog from rice,
OsGH3.13, is active on IAA, highly upregulated during
drought conditions and to a lesser extent, treatment with
the phytohormones IAA, SA and ABA, and confers
enhanced drought tolerance when overexpressed in rice
(Zhang et al. 2009).
For Group II, OsGH3.8 (in IIA) is upregulated following
infection with the bacterial pathogen Xanthomonas oryzae
pv oryzae and overexpresion of OsGH3.8 in rice is corre-
lated with increased levels of IAA-Asp conjugates and
enhanced resistance to Xanthomonas (Ding et al. 2008).
Similarly, constitutive expression of OsGH3.1 (in IIB)
alters auxin homeostasis and enhances resistance to the
fungal pathogen Magnaporthe grisea (Domingo et al.
2009). Furthermore, the IIA genes PtGH3.1 and PtGH3.2
were induced in EMF-colonized tree roots (Felten et al.
2009) and VvGH3.2 (probeset: 1610880_s_at) expression
was elevated in response to infection with Bois Noir phy-
toplasma (Albertazzi et al. 2009).
Discussion
GH3 phylogeny
A careful analysis of sequence data reconciled with syn-
tenic analysis indicates that GH3 phylogeny is not as clear-
cut as previously reported. Previous analyses of the GH3
gene family relied on global sequence similarities (Stas-
wick et al. 2005; Felten et al. 2009; Terol et al. 2006),
which are not necessarily indicative of true evolutionary
descent. The increasing number of sequenced genomes and
new comparative genomic tools makes it possible to use
synteny to evaluate phylogenetic trees. For example, Jun
et al. recently evaluated the use of local synteny for iden-
tifying orthologous genes in mammals, and found it quite
effective, particularly in cases of local duplication and
transposition (Jun et al. 2009). We used the syntenic data to
Induced Activity Phenotype
JA IAA SA JA IAA B JA IAA SA
Fig. 5 Maximum Likelihood phylogenetic tree of the ArabidopsisGH3 proteins showing induction by phytohormones, enzyme activity,
and mutant phenotype. Symbols in branches are as in Fig. 4. JA is
jasmonic acid, IAA is indole-3-acetic acid, SA is salicylic acid, B is
benzoates. In the ‘‘Induced’’ column, gray shaded boxes correspond
to C2-fold increase in expression compared to control treatment,
black shaded boxes correspond to C10-fold expression compared to
control treatment. Boxes with dashed line were not tested or did not
have gene specific data on array. PBS3 is highlighted. Microarray
data from NASCArrays set 174 (JA), 175 (IAA), 192 (SA) and 392
(SA-analogue BTH). Shaded boxes in the ‘‘Activity’’ column indicate
that the enzyme is active on the corresponding substrate. References
for activity data are (Staswick and Tiryaki 2004; Okrent et al. 2009).
Shaded boxes in the ‘‘Phenotype’’ column indicate that plants with
mutations in the genes have altered signaling through the phytohor-
mone indicated, with black shaded boxes moderate/strong and gray as
weak phenotype. References for phenotypic data are as follows:
AtGH3.1, (Staswick et al. 2005); AtGH3.2 (YDK1) (Staswick et al.
2005: Takase et al. 2004); AtGH3.5 (WES1) (Staswick et al. 2005;
Park et al. 2007b; Zhang et al. 2007); AtGH3.6 (DFL1) (Nakazawa
et al. 2001); AtGH3.9 (Khan and Stone 2007); AtGH3.11 (JAR1)
(Staswick et al. 1992); AtGH3.12 (PBS3) (Nobuta et al. 2007;
Jagadeeswaran et al. 2007; Lee et al. 2007); AtGH3.17 (Staswick
et al. 2005; Khan and Stone 2007)
Plant Mol Biol
123
guide the choice of phylogenetic methods in order to best
understand how PBS3 is related to the other GH3 genes.
Phylogenetic trees were constructed using neighbor-joining
(NJ), maximum parsimony (MP), and ML methods with
various alignment curation methods.
The ML method is a discrete data method that begins
with a model of rates of evolutionary change and alters the
model until in fits the observed data (Mount 2004). This is
contrasted with distance methods such as NJ for which the
percentage of aligned positions that differ between two
sequences is computed pair-wise for all sequences in an
alignment and the values arranged so that sequences with
lower difference scores are closer together on the tree
(Mount 2004). Distances methods tend to be favored by
molecular biologists as they are straightforward and com-
putationally efficient. The drawback of distance methods is
that they can be misleading when using an incorrect evo-
lutionary model (Huelsenbeck and Hillis 1993). While ML
methods are traditionally computationally intensive, new
algorithms, such as that employed by PhyML (Guindon
and Gascuel 2003) reduce calculation time sufficiently to
allow for routine use. There is considerable argument in the
literature over the ‘‘best’’ method for phylogenetic analysis,
often defined as how much a method can tolerate violations
of its assumptions (Huelsenbeck and Hillis 1993). For
example, there is a contentious debate over the relative
merits of ML particularly when different positions in a
sequence evolve over different rates (Steel 2005). Many
experts suggest constructing phylogenetic trees using
multiple methods and evaluating them carefully (Hall
2005; Mount 2004; Thornton and Kolaczkowski 2005).
When available, addition of data from syntenic analysis
can help determine the choice of phylogenetic method. We
found the tree constructed via ML best fit the data from
syntenic analysis and was supported by the available
functional data.
As shown in the eudicot GH3 ML phylogenetic tree in
Fig. 4, there are two clades with high statistical support. One
of these is Group I, which in turn forms two subclades: one
with JAR1 and its syntelogs (set IA) and one with DFL2 and
its syntelogs (set IB). Set IA predates the moncot/eudicot split
whereas set IB contains only eudicots (Online Resource 3).
As discussed in Results, JAR1 is active on JA and mediates
JA-dependent developmental and (a)biotic stress responses,
Pst
vir
Pst
avr
Psp
no
nh
ost
B. c
iner
a
P. i
nfe
stan
s
Ab
ioti
c st
ress
Induced
Bio
tic
Phenotype
Fig. 6 Maximum likelihood phylogenetic tree of Arabidopsis GH3
proteins and expression in response to biotic and abiotic stress.
Symbols in branches correspond to syntenic set as in Fig. 4. For the
‘‘Induced’’ column, black shaded boxes correspond to C2-fold
expression compared to control treatment and gray shaded boxescorrespond to C10-fold increase in expression compared to control
treatment at a = 0.05. Boxes with dashed line were not tested or did
not have gene-specific data on array. PBS3 is highlighted. Microarray
data is from adult leaves unless otherwise noted treated as indicated
from the following NASCArrays datasets: virulent, avirulent Pseu-domonas syringae pv. tomato and nonhost Pseudomonas syringae pv.
phaseicola (120), Botrytis cinerea (167), Phytopthora infestans (123),
seedlings treated with cold/osmotic/salt/drought (138–141). For the
‘‘Phenotype’’ column, shaded boxes indicate phenotypes have been
observed in mutants in response to biotic stress. Assessment of abiotic
stress response has been minimal so it has not been included here.
References are as follows: AtGH3.5 (WES1; Park et al. 2007a, b;
Zhang et al. 2007); AtGH3.6 (DFL1; Zhang et al. 2007); AtGH3.11
(JAR1; Ferrari et al. 2003); AtGH3.12 (PBS3, GDG1, WIN3; Nobuta
et al. 2007; Jagadeeswaran et al. 2007; Lee et al. 2007)
Plant Mol Biol
123
though it is not induced by JA nor (a)biotic stresses tested
(Figs. 5 and 6). Our analysis identifies JAR1 syntelogs in
agronomically important species including grape, poplar,
rice, and maize as potential targets for enhanced disease
resistance. By contrast, DFL2 is not active on JA or involved
in JA-associated responses (Figs. 5 and 6). Instead it mediates
red light-specific hypocotyl elongation with its expression
controlled by light (Takase et al. 2003, 2004) and its syntelogs
present only in the eudicots.
The second major clade contains the rest of the
sequences, which divides into two subclades, referred to as
Group II and Group III. Group II contains two major sets
(IIA and IIB) which precede the moncot/eudicot split. As
discussed in the Results, Group II members where tested
are induced by the growth phytohormone IAA, active on
IAA, with mutants exhibiting auxin-associated phenotypes
(e.g. Figs. 5 and 6). In addition, the promoters of the
Arabidopsis GH3 Group II member genes are enriched in
ARF- and ERF-binding motifs (Online Resource 4) con-
sistent with their induction by IAA and for some, by
pathogens (Figs. 5 and 6). Group II GH3 proteins in other
eudicot and moncot species are also induced by pathogen
and exhibit both auxin and altered susceptibility pheno-
types (see ‘‘Results’’).
Group III sequences consist of set IIIA (PBS3 and its
syntelogs, including AtGH3.17 which is active on IAA) and
set IIIB (AtGH3.9 which is active on IAA and its syntelogs).
As discussed earlier, AtGH3.9 and AtGH3.17 and their
orthologs had been previously classified as Group II proteins
though they did group separately from the other Group II
proteins (Staswick et al. 2005; Terol et al. 2006; Felten et al.
2009; Liu et al. 2005). Similar to Group II GH3 genes, Group
III genes are often induced by (a)biotic stress and altered
expression can result in (a)biotic stress phenotypes [i.e. At-
PBS3 (Nobuta et al. 2007; Lee et al. 2007; Jagadeeswaran
et al. 2007) and OsGH3.13 (Zhang et al. 2009)].
The most parsimonious explanation for the presence of
IAA- conjugating enzymes in both Group II and III is that
the ancestral gene encoded an enzyme that used IAA as a
substrate. Indeed, both rice Group II and III members
have been found to be active on IAA (Zhang et al. 2009;
Ding et al. 2008; Domingo et al. 2009). However, as
described in the Results, genes in both Group II and III
can be induced by SA and are active on benzoates [e.g.
Group II AtGH3.5 is active on SA (2-HBA) and Group III
PBS3/AtGH3.12 is active on 4-HBA]. IAA-amino acid
conjugates and their function in regulating auxin
homeostasis has been a subject of long-standing investi-
gation [reviewed in (Woodward and Bartel 2005)].
However, despite the fact that a variety of benzoate and
cinnamate amino acid conjugates have been detected in
plants (e.g. Suzuki et al. 1988; Bourne et al. 1991;
Trennheuser et al. 1994), GH3 protein substrate
preference has routinely been assessed only for
2-hydroxybenzoate (SA). Indeed, though PBS3 is not
active on SA, it is active on a series of other related
benzoates with a strong preference for 4-substituted ben-
zoates such as 4-HBA (Okrent et al. 2009). Additional
functional analyses of the Group III (and Group II) GH3
members including high throughput assays [described in
(Okrent et al. 2009)] to assess GH3 activity on a variety
of substrates combined with a comprehensive ancestral
analysis as more genomes are sequenced should eventu-
ally allow for a fuller understanding of the ancestral
function(s) and evolution of these proteins. Importantly,
the early emergence of GH3 proteins active on benzoates
is suggested by the ability of Lemna paucicostata (an
early diverging monocot) to produce benzoyl-Asp (Suzuki
et al. 1988) and the detection of 4-HBA-Glu and p-cou-
maroyl-Glu in the hornwort Anthoceros agrestis (Tren-
nheuser et al. 1994).
Function of PBS3 and its syntelogs
Contrary to previous reports that restrict PBS3 to Ara-
bidopsis and its close relatives, we identify PBS3 synte-
logs in poplar, grape, columbine, maize and rice
suggesting descent from a common ancestral chromosome
dating to before the eudicot/monocot split. Furthermore,
our analysis of co-linear genes found a syntenic relation-
ship between the Arabidopsis PBS3/AtGH3.12 gene and
AtGH3.17 that was obscured by sequence similarity alone.
The PBS3 syntelogs for which expression data exist are
induced by biotic interactions and/or abiotic stress, with
the exception of some of the recently acquired Arabidopis
genes (see ‘‘Results’’). As mentioned earlier, AtPBS3 is
active on 4-substituted benzoates while AtGH3.17 and
OsGH3.13 are active on IAA. However, these latter
enzymes were not tested for activity on 4-HBA. Loss of
PBS3 function alters benzoate metabolism, with a sub-
stantial impact on total SA accumulation and disease
resistance (Nobuta et al. 2007; Jagadeeswaran et al. 2007;
Lee et al. 2007). Enhanced expression of OsGH3.13/TLD1
alters plant architecture, IAA homeostasis, and enhances
drought tolerance (Zhang et al. 2009). However, a TLD1
loss of function mutant displayed no obvious growth or
drought phenotypes, presumably due to the compensatory
action of other GH3s (Zhang et al. 2009). Similarly, an
Arabidopsis gh3.17 mutant exhibited only very minor
auxin-associated phenotypes; it was not tested for altered
(a)biotic stress resistance (Staswick et al. 2005). Since
pbs3 mutants exhibit substantial defects in total SA
accumulation and disease resistance; benzoate metabolism
is likely to be the primary target of PBS3 and perhaps also
of its syntelogs. Cross-talk between SA, auxin, and the
drought-induced phytohormone ABA [e.g. (Park et al.
Plant Mol Biol
123
2007b; Zhang et al. 2009)], might then explain the phe-
notypes observed when OsGH3.13/TLD1 is overexpressed.
An examination of the predicted or known function of
conserved genes in the PBS3 syntenic regions (Online
Resource 2) may provide additional insight into the ancient
and perhaps conserved function of the PBS3 syntelogs.
Though benzoates are produced in the plastid, there is no
evidence that PBS3 is plastid-localized. However, many of
the genes in the PBS3 syntenic region are known or pre-
dicted to be in the plastid (Online Resource 2). NEF1
(At5g13390) syntelogs are present in most species exam-
ined including eudicots and monocots. NEF1 is plastid-
localized and involved in exine formation of pollen
(Ariizumi et al. 2004). The sculptured wall exine consists
of phenols and fatty acid derivatives and plays an important
role in protecting the pollen from various (a)biotic stresses
(in Ariizumi et al. 2004). Because NEF1 is expressed in
flowers, and benzoates including SA are known to impact
the induction of flowering (Martinez et al. 2004), pollina-
tion strategy (e.g. to act as specific pollinator attractants)
and defense (e.g. Dudareva and Pichersky 2000), we
examined whether PBS3 and the other Arabidopsis
genes in the PBS3 syntenic regions are expressed in
flowers. With the exception of the duplicated At5g13430
gene At5g13440, all Arabidopsis genes in the syntenic
region surrounding PBS3 are expressed in flowers (Online
Resource 2). Indeed, using a PBS3 promoter::GUS fusion,
Jagadeeswaran et al. (2007) found PBS3 to be expressed at
multiple stages of flower development and in specific floral
organs. Benzoate metabolism in flowers is integrated with
the functions of other phytohormones and can be modu-
lated by herbivory (e.g. see Kessler et al. 2010). Therefore,
a possible conserved function of PBS3 syntelogs may be to
modulate stress-induced benzoate metabolism in flowers
and by so doing provide a reproductive benefit.
In addition to its expression in flowers, NEF1 is strongly
induced by osmotic stress, heat, and cold in seedlings (abi-
otic stress, At-TAX series). Both PBS3 and OsGH3.13 are
also induced by drought/salt stress in addition to pathogens
and SA, as is the nearby ERF transcription factor At5g13330/
RAP2.6L (At-TAX dataset; Jagadeeswaran et al. 2007;
Zhang et al. 2009; Krishnaswamy et al. 2011). Similar to
overexpression of the PBS3 syntelog OsGH3.13 in rice
(Zhang et al. 2009), overexpression of RAP2.6L in Arabid-
opsis results in reduced stature, enhanced salt and drought
tolerance (Krishnaswamy et al. 2011). By contrast, rap2.6L
Arabidopsis mutants exhibit enhanced expression of SA-
dependent defense genes (e.g. PR1) and enhanced resistance
to Pseudomonas syringae (Sun et al. 2010), while pbs3
mutants are compromised in SA-dependent gene expression
and resistance to P. syringae (e.g. Nobuta et al. 2007).
Unfortunately, rice plants with altered OsGH3.13 expression
have not been tested for altered pathogen resistance/
susceptibility and Arabidopsis plants with altered PBS3
expression have not been tested for drought tolerance. In any
case, it is clear that PBS3 and its syntelogs can mediate
(a)biotic stress independent of flowering, with this role
supported by the (a)biotic stress-induction and, where tested,
function of its conserved neighbors NEF1 and RAP2.6L.
Expansion of the clade containing PBS3
in Arabidoposis
Of the rosids analyzed, A. thaliana was unique in that it not
only lost GH3 genes but also gained seven GH3 genes (Table
2). Intriguingly, the genes gained in Arabidopsis all are in
Group IIIA which contains PBS3. AtGH3.7 and AtGH3.8
were inserted into their location in the genome, as evidenced
by corresponding regions in papaya, grape, and poplar but no
GH3 gene (data not shown). AtGH3.13-16 (syntelogs of
PBS3) were locally duplicated, likely from the ancestral
gene of PBS3 as the proteins encoded by the four duplicated
genes are more similar to each other than they are to PBS3
(data not shown). The genes AtGH3.18 and AtGH3.19 rep-
resent a tandem duplication following insertion. All of these
rearrangements and insertions suggest a rapidly evolving
group of genes. Researchers have shown that genes that
respond to (a)biotic stress are overrepresented in arrays of
tandemly duplicated genes (Hanada et al. 2008; Rizzon et al.
2006) and tend to be retained following local duplication
events (reviewed in Freeling 2009). In addition, the same
genes that tend to be locally duplicated also tend to transpose
within chromosomal regions with high recombination rates.
Much of the work studying the divergence in expression of
duplicated genes has been done in yeast. However, expres-
sion patterns of tandemly and segmentally duplicated genes
in A. thaliana have been found to be similar, with a weak but
significant correlation between expression pattern and pro-
moter similarity (Haberer et al. 2004). The expression of the
tandemly duplicated sets of A. thaliana genes in the GH3
family, PBS3/AtGH3.12 and AtGH3.13-16; and AtGH3. 18
and AtGH3.19, do not retain correlated expression (data not
shown). The expression of the genes in different types of
tissues suggests possible specialization (Online Resource 5).
In addition, they differ in frequency of putative transcription
factor binding motifs (Online Resource 4), suggesting evi-
dence for promoter evolution. In many cases, these newer
AtGH3 genes are induced in response to (a)biotic stress
(Fig. 6) suggesting an evolving role for these genes in
response to stress. The rare effector HopW1-1 from Pseu-
domonas syringae pv. maculicola has been shown to bind to
the AtPBS3 protein (Lee et al. 2008) indicating unique tar-
geting of PBS3. This further illustrates the importance of the
PBS3 protein in disease resistance/susceptibility and sug-
gests host counter-evolution could play a role in the
expansion of this clade in Arabidopsis.
Plant Mol Biol
123
Acknowledgments We would like to thank Dr. Eric Lyons for his
assistance with the CoGe browser, Dr. Divya Chandran for careful
reading of the manuscript, and the William Carroll Smith Graduate
Research Fellowship in Plant Pathology (to R.A.O) and UC Berkeley
awards (to M.C.W.) for financial support. Some of the genome sequence
data described here was analyzed prior to publication by the sequencing
projects. Of these, the Aquilegia coerulea, Mimulus guttatus, and
Selaginella moellendorffii data were produced by the US Department of
Energy Joint Genome Institute. Carica papaya data were produced by
the ASGPB Hawaii Papaya Genome Project (http://www.
asgpb.mhpcc.hawaii.edu/papaya/). Zea mays data were produced by
the Genome Sequencing Center at Washington University School of
Medicine in St. Louis and can be obtained from http://www.
maizesequence.org/.
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