Evolution of the Aux/IAA Gene Family in Hexaploid Wheat

Abstract

The Aux/IAA (IAA) gene family, involved in the auxin signalling pathway, acts as an important regulator in plant growth and development. In this study, we explored the evolutionary trajectory of the IAA family in common wheat. The results showed ten pairs of paralogs among 34 TaIAA family members. Seven of the pairs might have undergone segmental duplication, and the other three pairs appear to have experienced tandem duplication. Except for TaIAA15-16, these duplication events occurred in the ancestral genomes before the divergence of Triticeae. After that point, two polyploidization events shaped the current TaIAA family consisting of three subgenomic copies. The structure or expression pattern of the TaIAA family begins to differentiate in the hexaploid genome, where TaIAAs in the D genome lost more genes (eight) and protein secondary structures (α1, α3 and β5) than did the other two genomes. Expression analysis showed that six members of the TaIAA family were not expressed, and members such as TaIAA8, 15, 16, 28 and 33 exhibited tissue-specific expression patterns. In addition, three of the ten pairs of paralogs (TaIAA5-12, TaIAA15-16 and TaIAA29-30) showed similar expression patterns, and another five paralog pairs displayed differential expression patterns. Phylogenetic analysis showed that paralog pairs with high rates of evolution (ω > ω 0), particularly TaIAA15-16 and TaIAA29-30, experienced greater motif loss, with only zero to two interacting IAA proteins. In contrast, most paralogous genes with low ω, such as TaIAA5-12, had more complete motifs and higher degrees of interaction with other family members.

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J Mol Evol (2017) 85:107–119
DOI 10.1007/s00239-017-9810-z
ORIGINAL ARTICLE
Evolution oftheAux/IAA Gene Family inHexaploid Wheat
LinyiQiao1,2· LiZhang3· XiaojunZhang2· LeiZhang4· XinLi2· JianzhongChang2·
HaixianZhan2· HuijuanGuo2· JunZheng2· ZhijianChang2
Received: 9 January 2017 / Accepted: 30 September 2017 / Published online: 30 October 2017
© Springer Science+Business Media, LLC 2017
Expression analysis showed that six members of the TaIAA
family were not expressed, and members such as TaIAA8, 15,
16, 28 and 33 exhibited tissue-specific expression patterns.
In addition, three of the ten pairs of paralogs (TaIAA5–12,
TaIAA15–16 and TaIAA29–30) showed similar expression
patterns, and another five paralog pairs displayed differential
expression patterns. Phylogenetic analysis showed that par-
alog pairs with high rates of evolution (ω > ω0), particularly
TaIAA15–16 and TaIAA29–30, experienced greater motif
loss, with only zero to two interacting IAA proteins. In con-
trast, most paralogous genes with low ω, such as TaIAA5–12,
had more complete motifs and higher degrees of interaction
with other family members.
Keywords Molecular evolution· Aux/IAA gene family·
Wheat· Expression analysis
Introduction
Whole-genome duplication (WGD) is a common event in plant
evolution and is of particular importance in improving the
growth and environmental adaptability of plants (Leitch and
Leitch 2008). Approximately 80% of angiosperms have expe-
rienced WGD events, and at least five WGD events occurred
in monocotyledons during their evolution (Masterson 1994;
Michael and VanBuren 2015), including many important poly-
ploidy crops such as the autopolyploid Chinese cabbage (Bras-
sica rapa, 2n = 4x = 38, AACC) and allopolyploid wheat (Triti-
cum aestivum, 2n = 6x = 42, AABBDD). The auxin signalling
pathway had a substantial impact on plant traits after the WGD
event (Paponov etal. 2009), as reported in many studies. For
example, in Chinese cabbage, auxin-related genes associated
with organ morphological variation expanded remarkably after
the WGD, which may be the cause of abundant morphological
Abstract The Aux/IAA (IAA) gene family, involved in the
auxin signalling pathway, acts as an important regulator in
plant growth and development. In this study, we explored
the evolutionary trajectory of the IAA family in common
wheat. The results showed ten pairs of paralogs among
34 TaIAA family members. Seven of the pairs might have
undergone segmental duplication, and the other three pairs
appear to have experienced tandem duplication. Except
for TaIAA15-16, these duplication events occurred in the
ancestral genomes before the divergence of Triticeae. After
that point, two polyploidization events shaped the current
TaIAA family consisting of three subgenomic copies. The
structure or expression pattern of the TaIAA family begins
to differentiate in the hexaploid genome, where TaIAAs in
the D genome lost more genes (eight) and protein secondary
structures (α1, α3 and β5) than did the other two genomes.
Electronic supplementary material The online version of this
article (doi:10.1007/s00239-017-9810-z) contains supplementary
material, which is available to authorized users.
* Jun Zheng
zhengjsxaas@126.com
* Zhijian Chang
czjsxaas@126.com
1 Department ofBiological Sciences, College ofLife Science,
Shanxi University, Taiyuan030006, China
2 Shanxi Key Laboratory ofCrop Genetics andMolecular
Improvement, Institute ofCrop Science, Shanxi Academy
ofAgricultural Sciences, Taiyuan030031, China
3 Department ofCrop Genetics andBreeding, College
ofAgriculture, Nanjing Agricultural University,
Nanjing210095, China
4 Department ofPlant Protection, College ofAgriculture,
Shanxi Agricultural University, Taigu030801, China

108 J Mol Evol (2017) 85:107–119
1 3
variations in the roots, stems and leaves (Brassica rapa
Genome Sequencing Project Consortium 2011). This varia-
tion revealed the crucial role of auxin pathway-related genes
in the growth, development and evolution of polyploid plants.
Common wheat (T. aestivum), a major staple crop, origi-
nated from the hybridization of the diploid Aegilops tauschii
(2n = 2x = 14, DD) and the ancestral tetraploid wheat T. turgi-
dum (2n = 2x = 28, AABB). The latter was formed via hybrid-
ization of T. urartu (2n = 2x = 14, AA) and Ae. speltoides
(2n = 2x = 14, SS ≈ BB) (Petersen etal. 2006; International
Wheat Genome Sequencing Consortium 2014). Brenchley
etal. (2012) found that many gene families were lost in the
genome of hexaploid wheat, while many other gene families
were greatly expanded at the same time. The expanded genes
included the auxin gene families related to plant growth and
metabolism. These changes are likely the key factors ena-
bling wheat to grow well in different areas and under com-
plex climatic conditions. Therefore, exploring the evolution of
auxin-related genes contributes to a better understanding of the
mechanisms underlying trait formation in wheat.
The Aux/IAA (IAA) family, which is typically involved
in the primary auxin response, consists of four functional
domains: Domains I–IV. IAA proteins can self-dimerize or
multimerize through Domains III and IV or combine with
the auxin response factor (ARF) to form a dimer that regu-
lates the downstream genes controlled by the auxin signal-
ling pathway. This signalling pathway can control growth
and development in plants (Dinesh etal. 2015), and it can
mediate interactions among the auxin pathway, light signal-
ling pathway (Halliday etal. 2009) and some hormonal sig-
nalling pathways, including those of brassinosteroids (Song
etal. 2009), ethylene (Strader etal. 2010), jasmonic acid
(Kazan and Manners 2009) and cytokinin (Schaller etal.
2015). Thus, the IAA family members are critical regulators
of the network controlling plant growth and development.
Thirty-four IAA family members (TaIAAs) with a total of
84 copies within three subgenomes were bioinformatically
isolated in our previous work from the hexaploid wheat cul-
tivar ‘Chinese Spring’ database (Qiao etal. 2015). Here, we
focused on the expression patterns of TaIAAs in different
tissues and organs of wheat and the evolution of this fam-
ily in wheat subgenomes and several plant species to better
understand the evolution process of polyploid species. Our
goal was to provide valuable information for further study
and utilization of TaIAAs in wheat.
Materials andMethods
Searching forIAA Family Sequences
The protein sequence data from Emiliania huxleyi, Physcom-
itrella patens, Selaginella moellendorffii and Brachypodium
distachyon were downloaded from the Joint Genome Insti-
tute Database (JGI, https://phytozome.jgi.doe.gov/). Pro-
tein sequence data for Picea abies and Pinus taeda were
downloaded from the Conifer Genome Integrative Explorer
(ConGenIE, http://congenie.org/), and protein sequence data
for Hordeum vulgare were downloaded from the Plant Tran-
scription Factor Database (PlantTF, http://lys.cbi.pku.edu.
cn:9010). The target amino acid sequences were obtained by
retrieving the protein database of each species using nHM-
MER software (Wheeler and Eddy 2013) with the IAA fam-
ily Hidden Markov Model profiles (Pfam accession number
PF00931) and E ≤ 1e−5. The IAA domains were checked
using the SMART server (Letunic etal. 2015) with default
values. The IAA protein sequences from the remaining spe-
cies were downloaded directly from the database according
to their registration numbers (TableS1).
Bioinformatics Analysis ofProtein Sequences
IAA consensus protein sequences of each species were
obtained by Jalview software (Waterhouse etal. 2009).
Multiple sequence alignments of these sequences were
performed using Clustal X (Larkin etal. 2007). Phyloge-
netic trees were constructed using the neighbour-joining
method in MEGA6.0 software (Tamura etal. 2013) with
1000 bootstraps. The MEME tool (Bailey etal. 2009) was
used to analyse IAA protein motifs in each species; the
length range of the motif was set to 8–50 amino acid resi-
dues and E ≤ 1e−100. The secondary structures of the pro-
tein sequences were predicted using the NPS server (Errami
etal. 2003). The STRING database (Szklarczyk etal. 2015)
was used to predict interactions among TaIAA members.
Synteny analysis was conducted in R using the 500-kbp
sequences both upstream and downstream of the TaIAA
genes (Ihaka and Gentleman 1996).
Calculation oftheEvolutionary Rate andDivergence
Time
As described by Koonin (2005), genes from different spe-
cies present on the same branch of the phylogenetic tree
were designated orthologs, while two TaIAA members on the
same branch with a high sequence similarity (> 30%) were
defined as paralogs. Guo’s method (Guo and Qiu 2013) was
adopted to define the paralogs located at different loci but
within the syntenic region of wheat chromosomes as seg-
mental duplication (SD), while paralogs located at the same
loci were defined as tandem duplication (TD) (Pont etal.
2013). The divergence times of TaIAA paralog pairs were
calculated using the formula T = Ks/2r (Li 1997), where the
divergence rate is 6.5 × 10− 9 (Gaut etal. 1996), and the Ks
(synonymous) and the evolution rates (ω) of different groups

109J Mol Evol (2017) 85:107–119
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in the phylogenetic tree were calculated by PAML software
(Yang 1997) with the codeml program.
Expression Analysis ofTaIAAs, TuIAAs andAetIAAs
The wheat cultivar ‘Chinese Spring’, used for tissue-specific
expression analysis, was planted with a long-day photo-
period (15-h light, 9-h darkness). The root, stem, flag leaf,
internode, leaf sheath and spikelet of every single plant were
harvested at the heading stage. Total RNA was extracted
using an RNA extraction kit (Tiangen Biotech, Beijing) and
reverse-transcribed into cDNA with an M-MLV reverse tran-
scription kit (Invitrogen, USA). RT-qPCR was performed
using SYBR Premix Ex Taq II (Takara Bio Inc., Dalian),
and each reaction was repeated three times with GADPH as
an internal control. RT-qPCR data were analysed using the
2−ΔΔCt
method (Livak and Schmittgen 2001). Primers listed
in TableS2 were designed to amplify all three genomic cop-
ies of each TaIAA member. Expression data for the AetIAA
family members were retrieved from the Aegilops tauschii
Goatgrass Genome Database (AGDB, http://dd.agrinome.
org/index.jsp), and expression data for the TuIAA fam-
ily were isolated from the transcriptome data, which were
downloaded from the Sequence Read Archive NCBI data-
base (https://www.ncbi.nlm.nih.gov/sra/?term=urartu).
Results
Aux/IAA Families inWheat andOther Plants
While no IAA proteins were isolated from Em. huxleyi,
there were 4, 8, 22 and 27 protein sequences identified
from Ph. patens, Se. moellendorffii, Pi. abies and Pi.
taeda, respectively. These protein sequences, together
with consensus sequences of wheat and other spe-
cies, were clustered into four subgroups (Groups I–IV)
(Fig.1a): Group I consisted of monocotyledons, in which
the IAA family of wheat A and D genomes showed the
closest genetic relationship to their ancestral species T.
urartu and Ae. Tauschii, respectively, followed by maize,
B. distachyon, rice and others. Group II showed a close
genetic relationship with the spermatophytes Arabidopsis
thaliana, Pi. taeda and others. Groups III and IV showed
a genetic relationship with pteridophytes and bryophytes.
The IAA family had relatively fewer members but longer
sequences in Ph. patens and Se. moellendorffii. Later,
the members began to grow in spermatophytes along
with multiple genome duplications (Michael and Van-
Buren 2015), and the sequences tended to be shorter and
more stable in angiosperms (Fig.1b). The IAA protein
sequences were relatively conserved during evolution; all
IAA proteins contain four functional domains (Domains
I–IV) and their signature sequences, including the amphi-
philic motif LxLxLx, which is necessary for transcrip-
tional repression in Domain I; VGWPP in Domain II,
which is the core sequence of the target site involved
Fig. 1 Aux/IAA families of wheat and another species. a Phyloge-
netic tree of Aux/IAA consensus protein sequences from six mono-
cotyledons (wheat, Ae. tauschii, T. urartu, B. distachyon, maize and
rice), two dicotyledons (A. thaliana and B. rapa), two gymnosperms
(Pi. taeda and Pi. abies), one pteridophyte (Se. moellendorffii) and
one bryophyte (Ph. patens). The WGD and whole-genome triplica-
tion are indicated by white and grey circles, respectively. b, c Num-
ber of members, average length of protein sequences and conserved
Domains I–IV of Aux/IAA family in each of these species. The α
structures are shown by a spiral, and β structure is shown by arrows.
The A, B and D genomes are identified by red, green and blue col-
ours, respectively. (Color figure online)

110 J Mol Evol (2017) 85:107–119
1 3
in the ubiquitin–proteasome pathway; and the α-helix
and β-folded structures associated with dimerization in
Domains III and IV (Nanao etal. 2014; Jing etal. 2015).
Moreover, the β1 structure was absent in genome A, while
α1, α3 and β5 structures were absent in the D genome of
wheat (Fig.1c).
Homologs ofTaIAAs inT. urartu andAe. tauschii
Phylogenetic trees were separately constructed with 34
TaIAAs (Fig.2) and with 34 TaIAAs (84 protein sequences
in total), 27 TuIAAs and 28 AetIAAs (Fig.S1). The TuIAA
and AetIAA homologs were found for 18 TaIAAs (TaIAA1,
3, 4, 9, 10, 11, 13, 18, 21, 22, 23, 24, 25, 28, 29, 30, 32 and
33), only AetIAA homologs were found for eight TaIAAs
(TaIAA2, 7, 8, 12, 17, 20, 26 and 34), while only TuIAA
homologs were found for five TaIAAs (TaIAA5, 6, 15, 19
and 27), and three TaIAAs (TaIAA14, 16 and 31) had no
homologous sequences (TableS3). TaIAA1, 5 and 28 all
had two TuIAA homologs, and TaIAA4 and 20 had two
homologous AetIAA sequences (TableS3). Therefore, these
homologous TuIAAs or AetIAAs may have duplicated after
the hexaploid wheat speciation. Alternatively, some of the
TaIAAs may have been lost in the wheat genome.
Duplication ofTaIAAs
There were 12 branches with TaIAA gene pairs in the phy-
logenetic tree (Fig.2). TaIAA15-B and TaIAA16-B, 1 of the
12 gene pairs, were located on the same chromosome 4BS
scaffold (Ensembl accession number: 4BS_scaffold330129).
Thus, TaIAA15 and 16 are paralogs from the formation of
TD. Moreover, TaIAA29 and 30 were both on chromosome
7S, while TaIAA29-D and 30-D were both located on chro-
mosome 7DS at 74.1cM (TableS4). It was assumed that the
TD of TaIAA29 and 30 occurred approximately 41.1MYA
(Table1) after the monocot–dicot divergence (130MYA,
D’Hont etal. 2012). Similarly, since TaIAA25-A and 26-A
were localized to a 53.81-cM region of chromosome 6AS
(TableS4), we hypothesized that they may also be TD
genes with a duplication time of approximately 17.8MYA
(Table1). In addition, no homologous genes for TaIAA25
and TaIAA26 were found in rice, maize and sorghum (Fig.2),
suggesting that the TD of TaIAA25 and 26 arose after the
divergence of rice (40MYA, Salse etal. 2008), maize and
Fig. 2 Phylogenetic tree and number of homologous genes of 34
TaIAA family members. a Phylogenetic tree is constructed by 84
TaIAA proteins. The TaIAA family can be categorized into Groups
A–O, and the branches of gene pairs are boldly represented. b Chro-
mosome position and the number of homologs in 14 species shown
in Fig.1 of TaIAAs. The A, B and D genomes of wheat, T. urartu and
Ae. tauschii are identified by red, green and blue colours, respectively.
(Color figure online)

111J Mol Evol (2017) 85:107–119
1 3
sorghum (20MYA, Salse etal. 2008). The remaining ten
gene pairs were in different genomic segments.
The synteny analysis was performed for each TaIAA pair
using a 500-kbp sequence both upstream and downstream
of each gene. Gene pairs TaIAA6 and 34 as well as TaIAA7
and 24 showed low sequence similarity (TableS4) and were
not in the syntenic segments of wheat subgenomes (Pont
etal. 2013). Therefore, we did not consider these pairs as
duplicated genes. Among the other seven gene pairs with
high sequence similarity (Fig.S2, Table1), TaIAA2 and 9,
TaIAA3 and 10, TaIAA4 and 13 and TaIAA5 and 12 were all
located in syntenic segments on chromosomes 1 through 3.
These pairs are probably SDs, as there were no homologs for
these four gene pairs in the dicotyledons, Gymnospermae,
Pteridophyta and Bryophytes listed in this study (Fig.2).
This finding suggested that the duplication likely occurred
in the ancestral genome of monocotyledons after the diver-
gence of monocotyledons and dicotyledons (130MYA,
D’Hont et al. 2012). Furthermore, gene pairs such as
TaIAA18 and 22 as well as TaIAA19 and 21 on chromo-
some sections 5S and 5L had similar SDs. The duplication
time of TaIAA11 and 32 on the chromosome sections 5S
and 7L was approximately 225.1MYA, suggesting that the
SD occurred in the ancestral genomes after the formation of
gymnosperms (350MYA, Nystedt etal. 2013). Furthermore,
TaIAA15 and 16 were located on the chromosome 4S–chro-
mosome 4L section with a sequence similarity of 39.85%.
Motifs andEvolution Rate ofTaIAAs
The TaIAA family can be divided into 15 groups (Group
A–O, Fig.3). The ω-analysis on each group indicated
that the sixteen-ratio model was predominantly superior
to the one-ratio model. Of the 15 selected evolutionary
branches with different evolution rates (TableS5), Group
M (containing paralogs TaIAA15 and 16) had the high-
est evolution rate (ω = 0.820), followed by Group J, which
included TaIAA29 and 30, with a ω of 0.200. Other groups
with high evolution rates (ω > ω0 = 0.140) were Groups
G, F and L. There were eight motifs in the TaIAA family
(Fig.3, TableS6). Motif 1, which belonged to Domain I,
was absent in TaIAA6, 8, 14, 16, 17, 25, 26 and 29; Motifs 2
and 3 belonged to Domain II, and both were absent in seven
members: TaIAA6, 8, 14, 16, 25, 26 and 31. All members
contained Motif 4, which belonged to Domain III; Motif 5
was a nuclear localization signal sequence (Abel and The-
ologis 1995), and a total of 11 members—TaIAA4, 5, 11, 12,
13, 18, 19, 22, 23, 27 and 32—contained Motif 5. Motifs 6,
7 and 8 belonged to Domain IV, and only nine members—
TaIAA3, 4, 10, 13, 18, 22, 23, 32 and 33—contained Motif
6. All members had Motif 7, and all members included Motif
8 except for TaIAA1, 14 and 27. In Group M, TaIAA16 con-
tained only Motifs 7 and 8, while TaIAA15-D lost Motifs 5,
6, 7 and 8. In Group J, TaIAA29 and 30 lost four to five and
three to four motifs, respectively.
Expression Patterns ofTaIAAs
The expression of 28 TaIAAs was detected in different tis-
sues and organs of wheat (Fig.4). Six members—TaIAA6,
7, 10, 17, 25 and 27—were not detected in the expression
data. Compared to other members, TaIAA28 exhibited the
highest expression in internodes and spikelets. The expres-
sion of TaIAA19 peaked in roots, stems and leaf sheaths.
TaIAA33 showed the highest expression in leaves (Fig.S3).
TaIAA8, 15, 16, 28 and 33 were highly expressed with tis-
sue-specific patterns. TaIAA8 was not expressed in roots,
leaves or spikelets, and TaIAA15 and 16 were not expressed
in leaves. No expression could be detected for TaIAA28 and
TaIAA33 in stems and spikelets, respectively (Fig.4). The
Table 1 Ten pairs of
paralogous TaIAAs in which
gene duplication occurs
MYA million years ago, SD segmental duplication, TD tandem duplication
a The duplication events are numbered according to the duplication time
Group Gene pairs Collinear section Similarity (%) Duplication
time (MYA)
Dupli-
cation
patterna
CTaIAA3–TaIAA10 Chro1S–Chro3S 37.45 90.32 #5 SD
ITaIAA2–TaIAA9 Chro1S–Chro3S 40.35 110.09 #4 SD
ITaIAA4–TaIAA13 Chro1L–Chro3L 42.3 85.42 #7 SD
DTaIAA5–TaIAA12 Chro1L–Chro3L 29.15 113.56 #3 SD
DTaIAA11–TaIAA32 Chro3S–Chro7L 41.6 225.12 #1 SD
ATaIAA18–TaIAA22 Chro5S–Chro5L 38.35 88.43 #6 SD
ETaIAA19–TaIAA21 Chro5S–Chro5L 37.5 114.78 #2 SD
JTaIAA29–TaIAA30 – – 41.11 #1 TD
LTaIAA25–TaIAA26 – – 17.82 #2 TD
MTaIAA15–TaIAA16 – – Uncertain #3 TD

112 J Mol Evol (2017) 85:107–119
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113J Mol Evol (2017) 85:107–119
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TuIAAs homologous to TaIAA6, 15, 22, 25, 29, 30 and 32
and the AetIAAs homologous to TaIAA7, 20, 29 and 30 were
not expressed (TableS3).
Of the ten pairs of paralogs, each of three gene pairs
(TaIAA5 and 12, TaIAA15 and 16, and TaIAA29 and 30)
showed similar expression patterns. Five gene pairs (TaIAA3
and 10, TaIAA25 and 26, TaIAA18 and 22, TaIAA19 and
21, and TaIAA11 and 32) had significant differences in
expression. TaIAA10 and 25 were not expressed as highly
as TaIAA18, 21 and 32. Furthermore, the expression data of
homologs for TaIAA15 and 16 and TaIAA29 and 30 were not
detected in T. urartu and Ae. tauschii, respectively.
Interaction Network Among TaIAA Proteins
The interaction between IAA proteins accelerated the speed
of the auxin response (Winkler etal. 2017). Twenty-eight
TaIAAs with expression data were used to carry out protein
interaction analysis. The results indicated that 15 members
were involved in interactions after the application of both
rice and B. distachyon interaction models (Fig.5). TaIAA18
and 22 from Group A; TaIAA5, 11, 12 and 32 from Group
D; TaIAA19 and 21 from Group E; TaIAA28 from Group
H; TaIAA4 and 13 from Group I; and TaIAA8 and 31 from
Group L all presented as high interaction, with the num-
ber of interacting proteins predicted to be 9–13 (TableS7).
However, TaIAAs from groups with high ω values, includ-
ing TaIAA15 and TaIAA16 from Group M (ω = 0.820),
TaIAA20 from Group G (ω = 0.188) and TaIAA7 and
TaIAA24 from Group F (ω = 0.173), were not predicted to
interact with other family members, except TaIAA29 and
TaIAA30 from Group J (ω = 0.200), which were predicted
to have only one to two interacting proteins.
In addition, interaction was usually predicted in some
important paralogs. In A. thaliana, the paralogs AtIAA6
and 19 interacted with each other in the ubiquitin degrada-
tion pathway to achieve a rapid response to auxin signal-
ling (Winkler etal. 2017). Similar interactions also exist
in rice paralogs OsIAA11–13 (Jing etal. 2015). Of the ten
pairs of paralogs in wheat, the members of only one pair,
TaIAA5–12, were predicted to interact with each other
under both the rice and B. distachyon interaction models
(Fig.5). Moreover, TaIAA5 and 12 had similar expression
patterns (Fig.4), and two wheat EST, sequences AK332471
and AK331670 (corresponding to TaIAA5 and TaIAA12,
respectively, in this study), were expressed in the absorp-
tion mechanism of wheat (Talboys etal. 2014). This finding
suggested that TaIAA5 and 12 are involved in the regulation
of auxin signalling pathways.
Discussion
Evolution oftheIAA Family inPlants
Auxin-related genes first emerged in primitive algae plants
(Cooke etal. 2002) as a simple structure. Subsequently,
the IAA–ARF auxin signalling pathway in bryophytes had
evolved to adapt to the terrestrial environment (Paponov
etal. 2009). The function of the auxin signalling pathway-
related genes began to differentiate in Pteridophyta, promot-
ing the formation of vascular bundles and realizing the rapid
transcription response to auxin (De Smet etal. 2011). Later,
in evolution, more relevant genes were involved in gymno-
sperms and angiosperms to regulate auxin to achieve more
advanced characteristics such as apical dominance and flow-
ering regulation (Huang etal. 2015; Priya and Ive 2013).
In the present study, the IAA family did not exist in the
phycophyte Em. huxleyi and was present as a few mem-
bers with long sequences in Bryophytes and Pteridophytes.
However, the IAA family had expanded in spermatophytes.
Although there was no evidence demonstrating that the gym-
nosperms Pi. abies and Pi. taeda had experienced WGD
events (Nystedt etal. 2013), the relatively large number of
members (27 and 22, respectively) in the PiaIAAs and Pit-
IAAs suggested an amplification of 12 ancient chromosomes
as a result of specific transposable element mobilization in
gymnosperms (Nystedt etal. 2013). The angiosperms under-
went several WGDs (Masterson 1994), WGD1 and WGD2
(Figs.1a, 6; 70–130MYA, Michael and VanBuren 2015),
subsequent to the divergence of monocotyledons, which
expanded the number of members in the IAA family to 27–34
in gramineous plants (Fig.1b).
In wheat, the TaIAA family may have experienced ten
gene duplications (seven SDs and three TDs), which all
occurred in the ancestral genomes predating the diver-
gence of Triticeae except TD #3. The first SD (225MYA)
occurred after the formation of angiosperms (350MYA,
Soltis etal. 2002; Nystedt etal. 2013), and the successive
SD #2–4 and SD #5–7 may have occurred following the
WGD1 and WGD2 events, respectively, in monocotyle-
dons. Two TDs occurred after the WGD3 (50–70 MYA,
Michael and VanBuren 2015). The abovementioned dupli-
cation events laid the foundation for the IAA family in
Triticeae. Thereafter, the TuIAA family and the AesIAA
family were integrated into the genome of T. turgidum
(WGD4, 0.8MYA, IWGSC 2014) via natural hybridiza-
tion. This was followed by hybridization with Ae. tauschii,
which introduced the AetIAA family into the hexaploid
wheat genome (WGD5, 0.4MYA, IWGSC 2014). After
Fig. 3 Phylogenetic tree and motifs of 84 TaIAA protein sequences.
The evolution rate (ω) of each group on the phylogenetic tree is
marked on the branch, and the asterisk refers to a ω value greater than
ω0 (0.140) in the sixteen-ratio model. Eight motifs of TaIAA proteins
were investigated with the MEME web server
◂

114 J Mol Evol (2017) 85:107–119
1 3
Fig. 4 Expression profiles of TaIAAs in different wheat organs. Total
RNA isolated from root (R), stem (ST), internode (I), leaf (L), sheath
(SH) and spikelet (SP) are used to assess the transcript levels of
TaIAAs in different wheat organs. Data represent the mean ± SD nor-
malized to the GADPH relative levels. All samples were run in tripli-
cate. Asterisks indicate no expression data

115J Mol Evol (2017) 85:107–119
1 3
several million years of natural and artificial selection in
the process of genome modification and expansion, the
TaIAA family in three subgenomes of common wheat was
eventually formed (Fig.6).
The number of members of the TaIAA family expanded
in WGD1, 2, 4 and 5, indicating that the gene duplication,
although it can randomly occur during the evolutionary pro-
cess, mostly occurred during major WGD events, which is
consistent with the findings of previous studies (Lynch and
Conery 2000; Raes and Van de Peer 2003). Of the TuIAA
genes homologous to TaIAAs-A, eleven were lost, four expe-
rienced gene duplication, at least one gene underwent chro-
mosomal translocation (Qiao etal. 2015) and eight members
were not expressed (Fig.6, TableS3). Of the AetIAA genes
homologous to TaIAAs-D, eight were lost, two were dupli-
cated and two underwent a chromosomal translocation (Qiao
etal. 2015), with five members not being expressed (Fig.6,
TableS3). These findings demonstrated that TaIAAs, TuIAAs
and AetIAAs had undergone different evolution processes
and exhibited different expression patterns.
Motif andStructure Changes During theEvolution
ofTaIAA
Polyploid plants are able to buffer against the shock brought
by genome doubling through gene recombination and modi-
fication, including chromosomal rearrangement, motif varia-
tion and gene expression regulation, along with the gradual
loss of a large number of redundant genes (Comai 2005;
Chen 2007; Otto 2007). Genes involved in basic physiologi-
cal functions and transcriptional regulation were retained to
maintain and stabilize the life activities of species (Freeling
and Thomas 2006; Birchler and Veitia 2007). Most poly-
ploid plants will gradually lose their polyploidy and even-
tually become diploid (palaeopolyploidy) during the evo-
lutionary process (Buggs etal. 2011; Roulin etal. 2013).
For instance, Chinese cabbage was originally hexaploid
before it lost one set of genes and became tetraploid (Bras-
sica rapa Genome Sequencing Project Consortium 2011).
A. thaliana, maize and soybean had been palaeopolyploid
(Roulin etal. 2013). Compared with these palaeopolyploids,
Fig. 5 Interaction networks of 28 TaIAA proteins expressed in wheat based on the interaction models of rice (a) and B. distachyon (b). Proteins
without interaction are not listed. The line thickness indicates the strength of the data support

116 J Mol Evol (2017) 85:107–119
1 3
Fig. 6 Evolutionary model of TaIAA gene family. The time of spe-
cies divergence and five WGDs on the axis in MYA is derived from
previous articles (Soltis et al. 2002; Yasumura et al. 2007; D’Hont
et al. 2012; Nystedt et al. 2013; Pont et al. 2013; IWGSC 2014;
Michael and VanBuren 2015), and nine gene duplication events
(seven SDs and two TDs) of the TaIAA family are marked in red.
Genomes from ancestors of grass and Triticeae are labelled in dark
grey, and the A, B and D genomes are identified by red, green and
blue colours, respectively. In the TuIAA, TaIAA and AetIAA families,
missing genes and non-expressed genes are displayed in white and
pale grey, respectively. (Color figure online)

117J Mol Evol (2017) 85:107–119
1 3
common wheat is a rather young allopolyploid in evolution.
Theoretically, each wheat gene has three copies of differ-
ent subgenomes referred to as ‘triplet genes’ (Pfeifer etal.
2014), whereas many studies have confirmed that substantial
sequence loss occurred during wheat polyploidization. In
light of research on synthetic hexaploid wheat (Liu etal.
1998; Zhang etal. 2014), low ploidy sequences in the D
genome were more likely to be lost during the polyploidi-
zation process. This phenomenon may be explained by the
tetraploid wheat (AABB), with a higher ploidy level, which
had undergone a polyploidization event during its evolu-
tion that resulted in the recombination and modification of
two genomes (AA and BB) to accommodate the impact of
allopolyploidization. In this study, TaIAAs-D had a higher
rate of gene loss (TaIAA-6D, 7D, 8D, 9D, 10D, 12D, 16D
and 31D) than those of TaIAAs-A and TaIAAs-B. The highest
rate of secondary structure loss (α1, α3 and β5) was found in
the consensus protein sequence of the remaining 26 TaIAAs-
D (Fig.1c), suggesting a massive sequence loss in the wheat
D genome during evolution.
In the phylogenetic tree of TaIAAs, the motif distribu-
tion and evolution rate were different in each group (Fig.3).
Motif loss was likely to occur in groups with a high ω value,
in which Groups M and F lost Motif 5+6, Group J lost Motif
2+3, while Motif 3+5+6 was lost in Group G and Motif
2+3+5+6 was lost in Group L (Fig.3). Furthermore, pre-
diction results showed that there was no or only one or two
interaction protein(s) in Groups F, G, M and J. This finding,
combined with the high ω value (Fig.5, TableS7), sug-
gested that these TaIAAs may obtain specific functions after
undergoing relaxed selective pressures. Of Group M, of the
highest ω value (ω = 0.82), TaIAA16 lost six motifs, leav-
ing only Motif 7+8, which belonged to Domain IV. Since
OsIAA6 significantly increased the tillering numbers of
transgenic plants after knocking out Motif 7+8 (Jung etal.
2015), it was inferred that TaIAA16 might also affect a spe-
cific developmental process. However, in groups with a low
ω value, most TaIAA motifs were comparatively complete
and possessed higher numbers of predicted interactions with
other family members (Fig.5, TableS7), such as Groups
A, B, D and I, which contained all eight classes of motifs.
Groups C and E contained seven classes (Fig.3), suggesting
that these TaIAAs maintained the most basic physiological
functions of plants and underwent relatively intensive selec-
tion. TaIAAs, especially TaIAA28 of Group H (ω = 0.014),
contained all homologous IAA sequences of 12 species
of seed plants (Fig.2), and the gene structure was similar
among these homologous IAA sequences (Fig. S4).
Expression Patterns ofthe TaIAA Family
Various expression patterns were detected in the TaIAA fam-
ily (Fig.4). Some of the 28 expressed TaIAAs were verified
by other experiments; for example, wheat EST sequences
AK332471 (corresponding to TaIAA5), AK331670 (cor-
responding to TaIAA12) and CK170519 (corresponding
to TaIAA21) exhibited differential expression during the
absorption of inorganic salts in wheat roots (Talboys etal.
2014). Moreover, in the present research, TaIAA5, 12 and
21 were in groups with low evolution rates, and the pre-
dicted results indicated that they were active in the interac-
tion network of TaIAA proteins, inferring an involvement
of these three genes in the basic nutritional physiological
processes of wheat. In addition, AJ575098 (correspond-
ing to TaIAA23) was expressed specifically in wheat tis-
sues and organs and hardly expressed at all in roots (Singla
etal. 2006). This finding was consistent with our research
(Fig.4). As with OsiIAA (Thakur etal. 2005), the expression
level of AJ575098 was also regulated by light (Singla etal.
2006). The study of the abovementioned TaIAAs will help
elucidate the molecular mechanism underlying wheat bio-
logical activities and provide a theoretical basis for tackling
practical production issues such as fertilizer utilization and
regulation of the growth period.
The expression data of TaIAA15–16 and TaIAA29–30
showed interesting results. Each of these two paralog pairs
had a high expression level and similar expression patterns
in different organs of wheat. However, their homologous
TuIAAs and AetIAAs were not expressed in T. urartu and
Ae. tauschii, respectively. In addition, compared with other
paralog pairs, both TaIAA15–16 and TaIAA29–30 had fewer
motifs (two to four) and interacting proteins (zero to two)
and belonged to the groups with the highest and second high-
est ω values, respectively. We hypothesized that the muta-
tions occurring after TD of these two paralogs bestowed
new traits in certain aspects of the plant. In wheat, these
new traits, which may conform to the needs of cultivation or
harvest, were preserved and abundantly expressed in plants
after a relaxed selection pressure. However, the variations in
T. urartu and Ae. tauschii were removed by purifying selec-
tion. In this study, only one pair of primers was designed
to analyse the expression of each TaIAA, while most genes
contained three copies within subgenomes of wheat. The
genome-specific primers are required to perform expression
analysis for different copies of each TaIAA member, since
only approximately 28% of the homologous triplet genes
exhibited the same expression pattern (Pfeifer etal. 2014).
Conclusions
The TaIAA family contains eight motifs, which can be fur-
ther divided into Groups A–O. The evolution rate of Group
M was 0.820, which is significantly higher than that of the
other groups. The rest of the groups with high ω values
were prone to motif loss and had almost no interaction

118 J Mol Evol (2017) 85:107–119
1 3
with other members. In contrast, motifs in groups with a
low ω value were relatively complete, possessed a higher
degree of interaction and may have experienced intensive
selection. Six members were not expressed, while the other
28 members were expressed. Eight had high expression
levels. Eleven genes exhibited relatively lower expression.
Genes with tissue-specific expression TaIAA8, 15, 16, 28
and 33 were detected at high expression levels. Among the
members of TaIAA family, TaIAA29–30 and TaIAA25–26
were likely to experience TD. TaIAA2–9, TaIAA3–10,
TaIAA4–13, TaIAA5–12, TaIAA15–16, TaIAA18–22,
TaIAA19–21 and TaIAA11–32 probably underwent
SD, which mainly occurred in the ancestral genome of
Gramineae 85–90 MYA and 110–114 MYA. For TaIAAs,
β1 structure loss was found in the consensus sequence of
the A genome, while genome D showed the loss of α1, α3
and β5 structures and eight TaIAA-D proteins.
Acknowledgements This study was funded by the National Key
Research and Development Plan of China (2016YFD0102004-07), the
National Natural Science Foundation of China (31601307), Shanxi
Province Science Foundation for Youths (2015021145), Shanxi Prov-
ince Natural Science Foundation (201601D102051) and Shanxi Prov-
ince International Cooperation Project (201603D421003). We thank
Dr. Xiaoyan Li (Beijing Anzhen Hospital Affiliated to the Capital
Medical University) and Dr. Wenping Zhang (Fujian Agriculture and
Forestry University) for their assistance in the RT-PCR experiment.
Compliance with Ethical Standards
Conflict of interest The authors declare that they have no conflict
of interest.
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