Genome-wide survey and comprehensive expression profiling of Aux/IAA gene family in chickpea and soybean

Abstract

Auxin plays a central role in many aspects of plant growth and development. Auxin/Indole-3-Acetic Acid (Aux/IAA) genes cooperate with several other components in the perception and signaling of plant hormone auxin. An investigation of chickpea and soybean genomes revealed 22 and 63 putative Aux/IAA genes, respectively. These genes were classified into six subfamilies on the basis of phylogenetic analysis. Among 63 soybean Aux/IAA genes, 57 (90.5%) were found to be duplicated via whole genome duplication (WGD)/segmental events. Transposed duplication played a significant role in tandem arrangements between the members of different subfamilies. Analysis of Ka/Ks ratio of duplicated Aux/IAA genes revealed purifying selection pressure with restricted functional divergence. Promoter sequence analysis revealed several cis-regulatory elements related to auxin, abscisic acid, desiccation, salt, seed, and endosperm, indicating their role in development and stress responses. Expression analysis of chickpea and soybean Aux/IAA genes in various tissues and stages of development demonstrated tissue/stage specific differential expression. In soybean, at least 16 paralog pairs, duplicated via WGD/segmental events, showed almost indistinguishable expression pattern, but eight pairs exhibited significantly diverse expression patterns. Under abiotic stress conditions, such as desiccation, salinity and/or cold, many Aux/IAA genes of chickpea and soybean revealed differential expression. qRT-PCR analysis confirmed the differential expression patterns of selected Aux/IAA genes in chickpea. The analyses presented here provide insights on putative roles of chickpea and soybean Aux/IAA genes and will facilitate elucidation of their precise functions during development and abiotic stress responses.

Full text

ORIGINAL RESEARCH
published: 27 October 2015
doi: 10.3389/fpls.2015.00918
Edited by:
Girdhar Kumar Pandey,
University of Delhi, India
Reviewed by:
Milind Ratnaparkhe,
Directorate of Soybean Research,
India
Haitao Shi,
Hainan University, China
*Correspondence:
Mukesh Jain
mjain@nipgr.ac.in
Specialty section:
This article was submitted to
Plant Physiology,
a section of the journal
Frontiers in Plant Science
Received: 20 July 2015
Accepted: 12 October 2015
Published: 27 October 2015
Citation:
Singh VK and Jain M (2015)
Genome-wide survey
and comprehensive expression
profiling of Aux/IAA gene family
in chickpea and soybean.
Front. Plant Sci. 6:918.
doi: 10.3389/fpls.2015.00918
Genome-wide survey and
comprehensive expression profiling
of Aux/IAA gene family in chickpea
and soybean
Vikash K. Singh and Mukesh Jain*
Functional and Applied Genomics Laborator y, National Institute of Plant Genome Research, New Delhi, India
Auxin plays a central role in many aspects of plant growth and development.
Auxin/Indole-3-Acetic Acid (Aux/IAA) genes cooperate with several other components
in the perception and signaling of plant hormone auxin. An investigation of chickpea
and soybean genomes revealed 22 and 63 putative Aux/IAA genes, respectively.
These genes were classified into six subfamilies on the basis of phylogenetic analysis.
Among63soybeanAux/IAA genes, 57 (90.5%) were found to be duplicated via
whole genome duplication (WGD)/segmental events. Transposed duplication played a
significant role in tandem arrangements between the members of different subfamilies.
Analysis of Ka/Ks ratio of duplicated Aux/IAA genes revealed purifying selection pressure
with restricted functional divergence. Promoter sequence analysis revealed several
cis-regulatory elements related to auxin, abscisic acid, desiccation, salt, seed, and
endosperm, indicating their role in development and stress responses. Expression
analysis of chickpea and soybean Aux/IAA genes in various tissues and stages of
development demonstrated tissue/stage specific differential expression. In soybean,
at least 16 paralog pairs, duplicated via WGD/segmental events, showed almost
indistinguishable expression pattern, but eight pairs exhibited significantly diverse
expression patterns. Under abiotic stress conditions, such as desiccation, salinity and/or
cold, many Aux/IAA genes of chickpea and soybean revealed differential expression.
qRT-PCR analysis confirmed the differential expression patterns of selected Aux/IAA
genes in chickpea. The analyses presented here provide insights on putative roles
of chickpea and soybean Aux/IAA genes and will facilitate elucidation of their precise
functions during development and abiotic stress responses.
Keywords: gene family, Aux/IAA, chickpea, soybean, gene duplication, transposed duplication, gene expression,
abiotic stress
INTRODUCTION
Auxin regulates cell division and elongation to drive plant growth and development (Woodward
and Bartel, 2005). Perception of auxin and control of auxin-regulated gene expression is
mediated by proteins belonging to three families including, receptors (F-box proteins), repressors
[Auxin/Indole-3-Acetic Acids (Aux/IAAs)] and transcription activators auxin response factors
(ARFs). The transmission of auxin signal depends upon interactions between components of these
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Singh and Jain Aux/IAA gene family in chickpea and soybean
protein families. Under low concentration of auxin, formation
of an ARF-Aux/IAA hetero-dimer results in the repression of
target ARF transcription factors (Tiwari et al., 2001;Guilfoyle and
Hagen, 2007). When auxin concentration is high, a co-receptor
complex consisting of an F-box protein from the transport
inhibitor response1 (TIR1)/auxin signaling F-box protein (AFB)
family and an Aux/IAA protein, binds auxin (Dharmasiri et al.,
2005;Tan et al., 2007). The F-box protein, being a component of
a Skp1–Cullin–F-box (SCF) E3 ubiquitin ligase (Gray et al., 2001;
Kepinski and Leyser, 2005), polyubiquitinylates and targets the
Aux/IAA proteins for degradation (Maraschin et al., 2009). This
degradation event relieves ARF transcription factor repression,
thus allowing auxin-regulated gene transcription (Reed, 2001;
Tiwari et al., 2001).
Aux/IAA proteins contain four conserved sequence motifs,
among which motif I, an amino-terminal leucine repeat motif
(LxLxLx) functions as transcriptional repressor of downstream
auxin-regulated genes (Tiwari et al., 2001, 2004). Motif II, a
TIR1/AFB recognition sequence with the conserved degron-
sequence, GWPPV, is responsible for the stability of Aux/IAA
proteins (Tiwari et al., 2004). Its interaction with the F-box
protein, TIR1, leads to rapid degradation of Aux/IAA proteins
(Dharmasiri et al., 2005). Motif III contains a βαα region and
motif IV represents an acidic region (Hagen and Guilfoyle, 2002;
Liscum and Reed, 2002). Motifs III and IV of Aux/IAA proteins
enable homo- and/or hetero-dimerization with other Aux/IAA
or ARF proteins and control the expression of downstream
auxin-responsive genes (Kim et al., 1997;Ulmasov et al., 1997;
Remington et al., 2004; Overvoorde et al., 2005). Although the
presence of four conserved motifs is characteristic of Aux/IAA
family, some members do not have one or more of these
motifs and are called non-canonical members (Reed, 2001;Jain
et al., 2006;Wang et al., 2010a;Audran-Delalande et al., 2012).
Particularly, some members lack conserved motif II and are
incapable of being recognized by TIR1/AFB proteins, indicating
that these Aux/IAA proteins may be involved in other auxin-
regulated biological processes (Jain et al., 2006;Kalluri et al.,
2007;Sato and Yamamoto, 2008;Wang et al., 2010a,b;Audran-
Delalande et al., 2012).
Many Aux/IAA genes have been characterized on the basis
of mutant analysis in Arabidopsis, which demonstrated the
important functions of Aux/IAA family genes in various
developmental processes. For example, functional loss of
IAA1/AXR5, a substrate of SCFTIR1, showed auxin-related
growth defects and auxin-insensitive phenotype (Ya n g et a l . ,
2004). Loss-of-function mutant, iaa3/shy2, affects auxin
homeostasis and formation of lateral roots (Uberti-Manassero
et al., 2012). The mutants, iaa7/axr2,iaa17/axr3,iaa19/msg2, and
iaa28 showed reduction in lateral root numbers (Tatematsu et al.,
2004;Okushima et al., 2007;Uehara et al., 2008;Rinaldi et al.,
2012), whereas iaa14/slr mutant blocked lateral root formation
entirely (Fukaki et al., 2002). A gain-of-function mutant, iaa16,
showed hampered plant growth and decreased response to auxin
(Rinaldi et al., 2012). In rice, over-expression of OsIAA1 led to
inhibition of root elongation and shoot growth (Song et al., 2009)
and a gain-of-function in OsIAA11 resulted in the loss of lateral
roots (Zhu et al., 2012). OsIAA23 was found to be involved in
post embryonic maintenance of quiescent center in rice (Jun
et al., 2011).
The members of Aux/IAA gene family have been identified
in several plant species, including Arabidopsis (Liscum and Reed,
2002), rice (Jain et al., 2006), Populus (Kalluri et al., 2007), maize
(Wang et al., 2010b), tomato (Wu et al., 2012), Vitis vinifera
(Cakir et al., 2013), and Medicago (Shen et al., 2014). However,
a genome-wide analysis of Aux/IAA gene family in chickpea and
soybean (for which genome sequences are available) is lacking
as of now. Chickpea and soybean are very important legume
crops, which serve as major source of proteins and carbohydrate.
Considering diverse role of Aux/IAA family members in other
plants, it is important to explore this gene family in chickpea
and soybean. In this study, we identified Aux/IAA genes in
chickpea and soybean genomes. We analyzed their sequence
characteristics, genomic organization, cis-regulatory elements,
and performed evolutionary duplication analysis. Furthermore,
we analyzed spatio-temporal differential expression between
Aux/IAA paralogs in various tissues/stages of development and
under abiotic stress conditions. These data would facilitate future
studies on elucidating the exact biological functions of Aux/IAA
genes in legumes.
MATERIALS AND METHODS
Identification of Aux/IAA Genes
Kabuli and desi chickpea genome annotations were downloaded
from Legume Information System1(LIS; Varshney et al., 2013)
and Chickpea Genome Analysis Project2(CGAP2; Parween et al.,
2015), respectively. Soybean genome annotation was downloaded
from Phytozome (v10, www.phytozome.net). Chickpea and
soybean proteomes were searched to identify Aux/IAA proteins
via HMMER and Basic Local Alignment Search Tool (BLASTP)
algorithms using the published Arabidopsis Aux/IAA protein
sequences as query. All obtained protein sequences were
examined for the presence of Aux/IAA (PF02309) domain
using the Hidden Markov Model of Pfam3and SMART4tools.
Physiochemical parameters of each gene were calculated using
ExPASy compute pI/Mw tool5. Information regarding cDNA
sequences, genomic sequences and ORF lengths were obtained
from the GFF file available at the respective genome project
webpages.
Gene Structure, Phylogenetic Analysis,
and Motif Prediction
Analysis of exon/intron organization of the Aux/IAA genes was
performed with Gene Structure Display Server6(GSDS). Multiple
sequence alignments of the full-length protein sequences from
chickpea, soybean, and Arabidopsis were performed with MAFFT
1http://cicar.comparative-legumes.org/
2http://nipgr.res.in/CGAP2/home.php
3http://pfam.sanger.ac.uk/search
4http://smart.embl-heidelberg.de
5http://web.expasy.org/compute_pi/
6http://gsds.cbi.pku.edu.cn
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Singh and Jain Aux/IAA gene family in chickpea and soybean
using default parameters and phylogenetic tree was constructed
by UPGMA method using CLC Genomics Workbench (v4.7.2).
Bootstrap analysis was performed using 1,000 replicates and the
tree was viewed using FigTree (v1.3.1). Motif organization of
chickpea and soybean Aux/IAA proteins was investigated by
MEME web server7.
Chromosomal Location and Gene
Duplication
Information about the chromosome location was obtained from
the GFF file and details of the segmentally duplicated regions in
the soybean genome were retrieved using the SyMAP database
(Soderlund et al., 2006). Synteny analysis for GmIAA genes was
performed using Plant Genome Duplication Database8(PGDD).
The genes and segmental duplicated regions were mapped to
the soybean chromosomes using the Circos tool (Krzywinski
et al., 2009). On the basis of Ks value obtained for each gene
pairs from PGDD, divergence time was calculated to investigate
evolution of soybean Aux/IAA genes.Thedivergencetime(T)
was calculated as T=Ks/(2 ×6.1 ×10−9)×10−6Mya,
based on a rate of 6.1 ×10−9substitutions per site per
year. For Ks value less than 0.3, divergence time was after
the Glycine whole genome duplication (WGD) event, when Ks
value was more than 1.3, divergence time was after the gamma
WGT (whole genome triplication) event, and if Ks value was
between 0.3 and 1.3, divergence time was after legume WGD
event and before the Glycine WGD event. To determine the
significance or contribution of the transposed duplication in
Aux/IAA gene evolution, Soytedb9was investigated to find out
nearest transposable elements around Aux/IAA genes.
Expression Profiling Using RNA-seq and
Microarray Data
For expression profiling in chickpea, we used the RNA-seq data
of 17 different tissues, namely germinating seedling (GS), root
(R), shoot (S), stem (ST), mature leaves (ML), young leaves
(YL), shoot apical meristem (SAM), flower bud stages (FB1-
4), flower stages (FL1-5), and young pod (YP) from previous
studies (Jain et al., 2013;Singh et al., 2013). High quality filtered
reads were mapped to the genome sequence of kabuli chickpea
(Varshney et al., 2013) using TopHat (v2.0.6). Cufflinks tool was
used to estimate the transcript abundance of genes in the form of
fragments per kilobase of transcript per million reads (FPKM) in
different tissues as described previously (Garg et al., 2015).
The expression of 63 GmIAA genes was investigated based
on the RNA-seq data from 19 tissues available at Gene
Expression Omnibus (GEO) database, including three samples
from soybean seed compartments, GloEP (globular stage embryo
proper; GSM721717), EmSCP (early maturation seed coat
parenchyma; GSM721719), and GloS (globular stage suspensor;
GSM721718); 10 other tissues samples, Gs (globular stage seed;
GSM721725), Hs (heart stage seed; GSM721726), Cs (cotyledon
7http://meme-suite.org/
8http://chibba.agtec.uga.edu/duplication
9http://www.soybase.org/soytedb
stage seed; GSM721727), Es (early maturation stage seed;
GSM721728), Ds (dry seed; GSM721729), R (root; GSM721731),
ST (stem; GSM721732), L (trifoliate leave; GSM721730), FB
(floral bud; GSM721733), and WS (whole seedling 6 days
after imbibition; GSM721734); three cotyledon development
samples, CoM (mid-maturation cotyledon; GSM721277), CoL
(late maturation cotyledon; GSM721278), and CoS (seedling
cotyledon; GSM721280); and three early maturation seed parts,
EcoEm (early maturation embryonic cotyledon; GSM1213-
856), EmEA (early maturation embryonic axis; GSM1213857),
and EmSC (early maturation seed coat; GSM1213855). For the
expression analysis of GmIAA genes, the RPKM method was
employed to correct for biases in total gene size and normalize for
total reads obtained in each tissue library (Mortazavi et al., 2008;
Nagalakshmi et al., 2008). Heatmaps of normalized expression
values of Aux/IAA genes of chickpea and soybean were generated
using R package pheatmap.
For abiotic stress response analysis of chickpea Aux/IAA
genes, we used raw RNA-seq data from root and shoot under
desiccation, salt and cold stresses from our previous study (Garg
et al., 2015). Read mapping and differential gene expression
analysis was performed as described (Garg et al., 2015)using
the kabuli chickpea genome as reference. The microarray data
of soybean under salt and drought stresses were downloaded
from the GEO database from accession numbers GSE41125 and
GSE40627, respectively. Probe sets corresponding to the GmIAA
genes were identified from the file GeneModels_AffyProbe.txt10.
Plant Materials, RNA Isolation and
Quantitative PCR Analysis
Chickpea (Cicer arietinum L. genotype ICC4958) seeds were
grown in field and culture room for collection of various tissue
samples. From field grown plants, mature leaf (ML), young
leaf (YL), flower buds (FB1-FB4; where FB1, FB2, FB3, and
FB4 were 4, 6, 8, and 8–10 mm size flower buds, respectively),
flowers (FL1–FL5; where FL1 was young flower with closed
petals, FL2 was flower with partially opened petals, FL3 was
mature flower with fully opened petals, FL4 was mature flower
with opened and faded petals and FL5 was drooped flower
with senescing petals), young pods (YP) were harvested as
described (Singh et al., 2013). Root (R), shoot (S), and GSs were
harvested as described (Garg et al., 2010;Singh et al., 2013).
Abiotic stress treatments (desiccation, salinity, and cold) were
given and root and shoot tissue were harvested as described
(Garg et al., 2010, 2015). Total RNA was isolated, quality, and
quantity were checked as described (Singh et al., 2015). Gene-
specific primers for selected CaIAA genes were designed using
the Primer Express (v3.0) software (Applied Biosystems, Foster
City, CA, USA) (Supplementary Table S1). Specificity of each
primer pair was determined via BLAST search. Quantitative
PCR reactions for at least two biological replicates each with
three technical replicates were performed employing 7500
fast real-time PCR system (Applied Biosystems) as previously
described (Garg et al., 2010). Elongation factor-1 alpha (EF-
1α) was used as a reference gene for normalization of gene
10http://www.seedgenenetwork.net/media
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Singh and Jain Aux/IAA gene family in chickpea and soybean
expression levels (Garg et al., 2010). Statistical significance of
the differential expression patterns was determined using the
Student’s t-test. Genes with ≥2-fold expression change (in at least
one tissue/condition/time point) with P≤0.05 were regarded as
differentially expressed.
Promoter Sequence Analysis
Genomic co-ordinates of coding sequences were determined
using GFF files obtained from chickpea and soybean genome
annotation projects. The regions of 1,000 bp upstream from start
codon were extracted from the genome sequences. Cis-regulatory
elements on both strands of promoter sequences were scanned
using NewPLACE web server11.
RESULTS AND DISCUSSION
Identification of Aux/IAA Genes in
Chickpea and Soybean
In order to identify the members of Aux/IAA gene family
in chickpea (kabuli) and soybean genome, BLASTP and
HMM profile searches were performed against their respective
proteomes. The Aux/IAA gene family members identified via
these two searches were combined and a non-redundant list was
obtained for chickpea and soybean. For further confirmation and
identification of the conserved Aux/IAA domains, all candidate
proteins were subjected to domain analysis using Pfam and
SMART databases. A total of 22 and 63 Aux/IAA genes in kabuli
chickpea and soybean genome, respectively, were confirmed.
Further the analysis of recent version of desi chickpea genome
(CGAP2) identified 21 Aux/IAA family members. BLASTP
analysis showed the presence of all these 21 Aux/IAA genes in the
kabuli chickpea genome. Due to identification of higher number
of genes, all further analyses were performed on Aux/IAA
genes from kabuli chickpea. Chickpea and soybean genes were
numbered according to their location on the chromosomes
(Supplementary Table S2). Various information of CaIAA and
GmIAA genes, including gene name, gene identifier, chromosome
location, mRNA length, features of deduced protein sequences,
and their gene, CDS, protein and promoter sequences are given
in Supplementary Table S2.
The number of CaIAA members (22) identified in chickpea
arelessascomparedtoArabidopsis (29; Liscum and Reed,
2002) and rice (31; Jain et al., 2006), but higher than its very
close relative Medicago (17; Shen et al., 2014). Lesser number
of Aux/IAA genes in chickpea and Medicago may be due to
some evolutionary constraints. However, the number of Aux/IAA
members in soybean (63) is much higher as compared to
other plants. Soybean possesses 9.2-fold larger genome size
(∼1,150 Mbp) and 1.75-fold higher gene count (∼46,400) than
Arabidopsis (Cannon and Shoemaker, 2012). Given the noticeable
differences in genome size and estimated gene count between
soybean and Arabidopsis,theAux/IAA genes in soybean seem
to be highly expanded. The presence of twice as many of these
11https://sogo.dna.affrc.go.jp/cgi-bin/sogo.cgi?sid=&lang=ja&pj=640&action=pa
ge&page=newplace
genes in soybean versus Arabidopsis maybemainlyduetothe
recent polyploidy and segmental duplication events in soybean
evolutionary history (Schmutz et al., 2010). The sizes of the
CaIAA proteins varied markedly ranging from 112 (CaIAA16) to
362 (CaIAA2) amino acids. Similarly, sizes of GmIAA proteins
also varied from 53 (GmIAA6) to 367 (GmIAA48) amino
acids. Furthermore, predicted isoelectric points varied from 4.64
(CaIAA19) to 9.63 (CaIAA1) in chickpea and 5.24 (GmIAA42) to
9.27 (GmIAA25) in soybean, suggesting that different CaIAA and
GmIAA proteins might function in different microenvironments.
Phylogenetic Relationship, Gene
Structure and Sequence Similarity
To examine the phylogenetic relationship among the Aux/IAA
proteins of chickpea, soybean, and Arabidopsis, a rooted tree
was constructed using alignments of their full-length amino-acid
sequences (Figure 1). Phylogenetic distribution indicated that
Aux/IAA proteins can be classified into two major groups, A
and B (Figure 1) similar to Arabidopsis and rice (Remington
et al., 2004;Jain et al., 2006), which are further subdivided
into four and two subgroups, respectively. Similar groupings
have been reported in other plant species too (Cakir et al.,
2013;Gan et al., 2013). The group A (A1–A4) consisted of 12
members of CaIAA and 41 GmIAA proteins, structuring 25
sister pairs (five pairs of GmIAA-CaIAA, 16 pairs of GmIAA-
GmIAA and four pairs of AtIAA-AtIAA proteins). Group B
(B1–B2) included 10 CaIAA and 22 GmIAA proteins, which
formed 15 sister pairs (11 pairs of GmIAA–GmIAA, four
pairs of AtIAA–AtIAA). Phylogenetic tree topology revealed
that sister pairs located at the terminal nodes show high
similarity and were assigned as paralog or ortholog pairs
(Figure 1, Supplementary Figure S1). The sequence similarity
within chickpea and soybean Aux/IAA proteins ranged from
9 to 80.3 and 6.5 to 93.9%, respectively (Supplementary
Figure S1). All paralog pairs of soybean determined through
phylogenetic analysis were found to be duplicated via WGD
events (Figure 1, Supplementary Table S3), except GmIAA6
and 7 (tandemly duplicated). Furthermore, higher sequence
similarity was observed between paralog pairs, suggesting that
these genes evolved via genome duplication event and may
perform similar functions. Interestingly, phylogenetic analysis
predicted four homologs of AtIAA16 in the soybean genome
(GmIAA14, 36, 59, and 63). In Populus, four orthologs of
AtIAA16 have also been found, but were absent in rice,
indicating their specific function in dicots. Moreover, diversity
of gene structure (exon-intron organization) is also a possible
explanation for the evolution of multigene families. The exon-
intron organization in the coding sequences of each Aux/IAA
genes of chickpea and soybean were compared. As expected,
in most of the sister-pairs, similar exon-intron organization
was observed. This conservation of exon-intron organization
between subfamilies and the dissimilarity within subfamilies
supported the results of phylogenetic and genome duplication
analysis.
The established model for auxin signal transduction
represents auxin-mediated degradation of these short-lived
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Singh and Jain Aux/IAA gene family in chickpea and soybean
FIGURE 1 | Phylogenetic relationship among Aux/IAA genes from chickpea, soybean, and Arabidopsis.The deduced full-length amino acid sequences of
chickpea (CaIAA), soybean (GmIAA), and Arabidopsis (AtIAA) genes were aligned by MAFFT and the phylogenetic tree was constructed by CLC Genomics
Workbench using the UPGMA method. The members of each Aux/IAA subfamily are shown in different colors. The numbers on the nodes represent bootstrap
values from 1000 replicates.
proteins that have four characteristic conserved domains.
Conspicuously, the chickpea genome represents six such non-
canonical Aux/IAA proteins (CaIAA5, 11, 12, 16, 17, and 19) that
do not have conserved domain II, which is crucial for protein
degradation, whereas 13 (GmIAA5, 6, 13, 23, 31, 35, 37, 39, 40,
42, 53, 58, and 60) such proteins were found in the soybean
genome (Figure 2). These non-canonical proteins were found to
be long-lived as compared to the canonical Aux/IAA proteins
(Dreher et al., 2006). In tomato, such non-canonical Aux/IAA
proteins were found to have expression pattern restricted to
narrow development stages (Audran-Delalande et al., 2012),
suggesting that these proteins may have a very specific function
during development in plants for mediating auxin responses.
Chromosomal Location and Duplication
The chromosomal distribution of 22 CaIAA genes revealed their
location on all the eight linkage groups (Supplementary Table
S2). Eight CaIAAs were present on chromosome 4, five on
chromosome 7, three on chromosome 3, two on chromosome
6, and one on chromosome 1, 2, 5, and 8 each. In soybean, 63
GmIAA genes were located on 16 of 20 chromosomes, except
for chromosomes 11, 12, 16, and 18 (Figure 3, Supplementary
Table S2). Out of 63 GmIAA genes, nine genes were present on
chromosome 10, eight on chromosome 13, six on chromosome 2,
five on chromosome 3, 8, and 19 each, four on chromosome 7, 15,
and 20 each, three on chromosome 1, and two on chromosome
4, 6, 9, and 17 each. Chromosomes 5 and 14 harbored only
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Singh and Jain Aux/IAA gene family in chickpea and soybean
FIGURE 2 | Gene structure and motif organization of Aux/IAA family members in chickpea and soybean. Left panel illustrates the exon–intron organization
of Aux/IAA genes in chickpea and soybean. The exons and introns are represented by boxes and lines, respectively. Right panel shows motif organization in
chickpea and soybean Aux/IAA proteins. Motifs of Aux/IAA proteins were investigated by MEME web server. Six motifs representing four domains I, II, III, and IV of
Aux/IAA proteins are displayed at the bottom.
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Singh and Jain Aux/IAA gene family in chickpea and soybean
FIGURE 3 | Mapping of segmentally duplicated GmIAA genes on soybean chromosomes. Gray ribbons indicate collinear relationships among the blocks in
whole genome and blue ribbons show GmIAA paralogs. The soybean chromosomes are arranged in a red circle and the size of each arc show the size of respective
chromosome (Mb).
one GmIAA gene each. The chromosomal location of Aux/IAA
genes of chickpea and soybean showed tandemly located gene
clusters. The gene cluster in chickpea included CaIAA3 and 4on
chromosome 3. For soybean, eight such clusters were observed,
including GmIAA6,7and 8on chromosome 2, GmIAA10,
and 11 on chromosome 3, GmIAA32 and 33,GmIAA37 and
38 on chromosome 10, GmIAA46 and 47 on chromosome 13,
GmIAA49 and 50 on chromosome 15, GmIAA55 and 56 on
chromosome 19, and GmIAA61 and 62 on chromosome 20.
Soybean genome has undergone one WGT and two WGD
events (legume WGD and Glycine WGD), and about 75%
genes have multiple paralogs (Schmutz et al., 2010;Severin
et al., 2011). Among paralog genes, ∼50% displayed expression
subfunctionalization (Roulin et al., 2012)thatmaycause
phenotypic variation in polyploids (Buggs et al., 2010). Besides
WGD, tandem duplication generates consecutive gene copies
in the genome through unequal chromosomal crossing over
(Freeling, 2009) and may contribute in the expansion of
gene families (Cannon et al., 2004). Dispersed duplicates
(not tandemly or segmentally duplicated) arises via either
DNA or RNA based transposition mechanisms (Ganko et al.,
2007;Cusack and Wolfe, 2007;Freeling, 2009)andmayplay
an important role in altering gene function and creating
new genes (Woodhouse et al., 2010;Wang et al., 2011).
To find the potential relationship between putative paralog
pairs of Aux/IAA genes of soybean and tandem/segmental
duplications, we performed duplication analysis using PGDD.
Within the identified duplicated GmIAAs, a larger fraction of
them (57, 90.47%) were duplicated through WGD/segmental
events, and only GmIAA6 and 7were tandemly duplicated
(Figure 3, Supplementary Table S3). In the syntenic block,
some genes from different subfamilies showed the tandem
relationship. For example, paralog gene pairs, GmIAA6/33,
GmIAA10/55,GmIAA37/62, and GmIAA46/50 displayed tandem
relationship with GmIAA8/32,GmIAA11/56,GmIAA38/61, and
GmIAA47/49, respectively (Figure 3, Supplementary Tables S2
and S3). Presence of transposable elements in the flanking regions
of these genes, suggested that they were tandemly arranged
due to transposed duplication events (Supplementary Table
S4). In addition to WGD events, some other gene duplication
events were also found in few Aux/IAA genes of soybean.
In A1 subfamily, six paralog genes (GmIAA8/32,10/55, and
37/62) resulted from the common ancestor sites experiencing
three WGD events, while a dispersed gene (GmIAA4) through
the transposed duplication located among two transposable
elements (Supplementary Tables S3 and S4). In A4 subfamily,
ten paralog genes (GmIAA6/33,11/56,14/59,36/63, and
38/61) were resultant from common ancestor sites, which
Frontiers in Plant Science | www.frontiersin.org 7October 2015 | Volume 6 | Article 918

Singh and Jain Aux/IAA gene family in chickpea and soybean
experienced three WGD events, while GmIAA7 was found to
have tandem connection with GmIAA6 (Supplementary Tables
S3 and S4). The GmIAA genes grouped into B1 and B2
subfamilies exhibited transposed duplication, which were flanked
by two transposable elements each (Supplementary Tables S3
and S4).
Furthermore, to investigate whether Darwinian positive
selection is involved in the divergence of GmIAA genes
after duplication and to trace the dates of the duplication
blocks, the substitution rate ratios (Ka/Ks) of all paralog
pairs were extracted from PGDD database. Ksvalueswere
used for calculating approximate dates of duplication events.
The segmental duplications of the GmIAA genes in soybean
were assumed to originate from 2.35 Mya (million years ago,
Ks=0.03) to 327 Mya (Ks=4.26), with a mean value
of 17.6 Mya (Ks=0.23, Supplementary Table S5). Previous
studies have shown that the soybean genome has undergone
two rounds of WGD, including an ancient duplication prior
to the divergence of papilionoid (58–60 Mya) and a Glycine-
specific duplication that has been estimated to have occurred
∼13 Mya (Schmutz et al., 2010). Most of the WGD/segmental
duplications of the GmIAA genes seem to have occurred
around 13 Mya when Glycine-specific duplication occurred
(Supplementary Table S5). According to the ratio of non-
synonymous to synonymous substitutions (Ka/Ks), the history
of selection acting on coding sequences can be measured (Li
et al., 1981). A pair of sequences will have Ka/Ks <1, if one
sequence has been under purifying selection, but the other has
been drifting neutrally, while Ka/Ks =1, if both the sequences
are drifting neutrally and rarely, while Ka/Ks >1atspecific
sites, when they were under positive selection (Juretic et al.,
2005). Ka/Ks for all GmIAA duplicated pairs were less than 1
(Supplementary Table S3), which suggests that all gene pairs
have evolved mainly under the influence of purifying selection
pressure with limited functional divergence after segmental
duplications.
Cis-regulatory Elements in Promoters of
CaIAAs and GmIAAs
The analysis of cis-regulatory elements in the promoter sequences
is an important aspect in understanding the gene function
and regulation. We searched 1 kb promoter region of all the
CaIAA and GmIAA genes to determine putative cis-regulatory
elements involved in their transcriptional regulation using
NewPLACE database. Many cis-regulatory elements identified
in the promoters were found to be related to auxin, ABA, SA,
sugar, light, drought, salt, and cold responses indicating that these
genes are linked to phytohormone signals, and/or abiotic stresses
(Supplementary Table S6). Previous studies suggest that light is
involved in regulation of Aux/IAA protein activity. For example,
phytochrome A (phyA) interacts with Aux/IAA proteins, as
revealed by yeast two-hybrid analysis (Soh et al., 1999). Moreover,
oat phyA was able to phosphorylate IAA1, SHY2/IAA3, IAA9,
AXR3/IAA17, and PS-IAA in vitro (Abel et al., 1994;Soh et al.,
1999). The domain II mutants, axr2-1,axr3-1,shy2-1,andshy2-2,
develop leaves in dark (Kim et al., 1998;Reed et al., 1998;Nagpal
et al., 2000). Presence of auxin, ABA and cytokinin responsive
cis-regulatory elements in the promoters of CaIAA and GmIAA is
also consistent with previous reports, such as the interactions of
auxin with other phytohormones (cytokinin or ABA) to regulate
many aspects of plant growth and development (Paponov
et al., 2008). Many CaIAA and GmIAA genes were found to
harbor AuxRE motif in their promoter, which is important for
binding of ARFs and transcriptional activation of Aux/IAA genes
(Tiwari et al., 2001, 2003). Interestingly, promoter sequences
of those CaIAA and GmIAA genes, which lack AuxRE motif,
were found to harbor sugar-responsive motif (SREATMSD),
suggesting that there is an association between sugar and auxin
responses. Both sugar and auxin are essential for plants and
control similar processes. In Arabidopsis, these two signaling
pathways were found to interact with each other (Mishra
et al., 2009). Bioactive GAs (gibberellic acid) influence nearly
all aspects of plant growth and development from germination
to hypocotyl elongation, stem growth, circadian rhythm, and
reproductive organ and seed development (Lovegrove and
Hooley, 2000;Hartweck and Olszewski, 2006). The presence
of gibberellic acid response element (GARE) in many of the
Aux/IAA genes (Supplementary Table S6), indicate their role
in such processes. In addition, cis-regulatory elements known
for regulation of endosperm, embryo, cotyledon, seed storage
proteins related responses were also predicted in the CaIAA and
GmIAA gene promoters (Supplementary Table S6), suggesting
their role in seed development. Circadian element, which is
involved in circadian control, was abundantly found in the
promoter region of chickpea and soybean Aux/IAA genes
(Supplementary Table S6), potentially indicating that they may
have a distinct diurnal expression pattern. In Arabidopsis and
rice, a class of element defined by the core motif ‘(a/g)CCGAC’
named as dehydration responsive element/C-repeat (DRE/CRT)
was reported for drought, low temperature, and salt inducible
expression (Yamaguchi-Shinozaki and Shinozaki, 1994;Meier
et al., 2008). Interestingly, we also found this motif in many
of the CaIAA and GmIAA gene promoters (Supplementary
Table S6), indicating their role under abiotic stress conditions.
Overall, the promoter analysis demonstrated the presence of a
variety of cis-regulatory elements in the upstream regions of
chickpea and soybean Aux/IAA genes. These results provide
further support for the various functional roles of Aux/IAA genes
in a wide range of developmental processes and abiotic stress
responses.
Differential Expression of Chickpea and
Soybean Aux/IAA Genes during
Development
ToknowtheputativefunctionofAux/IAA genes in chickpea
and soybean during development, we analyzed their expression
profiles in different vegetative and reproductive tissues, using
available RNA-seq data sets (Jain et al., 2013;Singh et al.,
2013). Chickpea RNA-seq data included eight tissues/organs,
such as GS, root (R), shoot (S), stem (ST), mature leaf
(ML), young leaf (YL), SAM, young pod (YP) and nine
stages of flower development (FB1-4 and FL1-5). Many of
CaIAAs illustrated a distinct tissue-specific expression pattern
Frontiers in Plant Science | www.frontiersin.org 8October 2015 | Volume 6 | Article 918

Singh and Jain Aux/IAA gene family in chickpea and soybean
FIGURE 4 | Expression profiles of chickpea Aux/IAA genes. (A) The normalized RNA-seq expression data was used to construct heatmap. Hierarchical
clustering was conducted in R using the pheatmap package with a dissimilarity metrics based on Euclidean distance with complete linkage algorithm. Color key at
the bottom represents row wise Z-score. (B) qRT–PCR analysis of CaIAA genes in various tissue/stages of development. Expression of germinating seedling (GS)
was taken as a reference to determine relative mRNA level in other tissues for each gene. Error bars indicate SE of mean. R (root), S (shoot), ST (stem) ML (mature
leaf), SAM (shoot apical meristem), FB1-4 (stages of flower bud), FL1-5 (stages of flower), YP (young pod). Data points marked with asterisk (∗P≤0.05, ∗∗P≤0.01,
and ∗∗∗P≤0.001) indicates statistically significant difference between control (GS) and other tissues.
(Figures 4A,B). For example, CaIAA15 and 16 revealed specific
expression in stem, indicating their role in stem development
(Figures 4A,B; Supplementary Table S7). It has been found
that mutation in Aux/IAA genes affect stem elongation (Reed,
2001). Furthermore, CaIAA1, 3, 11, and 12 showed higher
expression in SAM, among which CaIAA3 was validated through
qRT-PCR (Figures 4A,B; Supplementary Table S7), implying
their involvement in SAM maintenance. In rice, OsIAA23
was found to be involved in postembryonic maintenance of
quiescent center (Jun et al., 2011). CaIAA4, 7, 10, 13, and
21 revealed higher transcript accumulation during stages of
flower development (Figures 4A,B; Supplementary Table S7),
suggesting their possible role in flower development. In rice,
OsIAA4 and 26 were found to be up-regulated in panicle
(Jain and Khurana, 2009), while MtIAA9 in Medicago, showed
higher expression level in flower (Shen et al., 2014). CaIAA14,
17 and 18 revealed distinctly higher expression in stages of
flower development and young pod (YP). In tomato, SlIAA9
was shown to be involved in fruit development (Wang et al.,
2005). The expression profile of at least four CaIAA (CaIAA3,
16,18, and 21) genes was studied by qRT-PCR to validate the
RNA-seq results. The expression patterns obtained via qRT-
PCR were found to be well correlated with that of RNA-seq
(Figures 4A,B).
For soybean, normalized RNA-seq data from 19 tissues were
used, which included various tissues/organs, seed compartments,
and stages of seed development. GmIAA genes showed specific
and overlapping expression patterns in various tissues/organs
and stages of development analyzed (Figure 5, Supplementary
Table S7), indicating they might execute specific functions
or work redundantly. GmIAA60 was specifically expressed in
WS (whole seedling), while its paralog GmIAA39 expressed
specifically in Ds (dry seed), indicating their functional
divergence. GmIAA15 has higher expression in root (Figure 5,
Supplementary Table S7) and presence of root-responsive
cis-regulatory element in its promoter (Supplementary Table
S6), suggested its role in root development. In Arabidopsis,
many IAA genes (AtIAA1,AtIAA18, and AtIAA28)werealso
found to have role in root development (Rogg et al., 2001;
Yang et al., 2004;Uehara et al., 2008). In addition, paralogs
GmIAA45 and 51 showed higher transcript accumulation in
shoot (Figure 5, Supplementary Table S7), signifying their
functional conservation with role in shoot development, which
is further revealed by presence of shoot responsive cis-
regulatory element in their promoter sequences (Supplementary
Table S6). However, GmIAA6,31, and 33 exhibited higher
transcript levels in floral bud (FB) (Figure 5, Supplementary
Table S7) and detection of pollen specific cis-regulatory
elements in their promoter sequences suggests their putative
role in development of FBs. Many GmIAA genes were
detected with increased transcript accumulation at various
stages/organs of seed development too. For instance, GmIAA49
exhibiting specific expression in Gs (globular stage seed),
GmIAA13, 35, 43, 54, and 56 in GloEP (globular stage embryo
proper), GmIAA5 in GloS (globular stage suspensor), GmIAA22
and GmIAA23 in Cs (cotyledon stage seed), GmIAA59 in
EmSCP (early maturation seed coat parenchyma), GmIAA62
in EmEA (early maturation embryonic axis), and GmIAA42
in CoL (late maturation cotyledon) (Figure 5, Supplementary
TableS7).Inaddition,manyseedrelatedcis-regulatory
elements were detected in their promoter sequences, such
as S000143, S000353, S000449, S000148, S000421, S000292,
S000144, S000419, S000420, S000100, S000102, and S000377
(Supplementary Table S6), that further added support for their
putative role in seed development. In Arabidopsis, two Aux/IAA
proteins have also been reported for their involvement in seed
development (Hamann et al., 1999;Ploense et al., 2009). In
the gain-of-function mutant, iaa18, PIN1 was asymmetrically
expressed with stronger expression at only one side of the
embryo and caused aberrant cotyledon outgrowth in the
Frontiers in Plant Science | www.frontiersin.org 9October 2015 | Volume 6 | Article 918

Singh and Jain Aux/IAA gene family in chickpea and soybean
FIGURE 5 | Expression profiles and the evolutionary patterns of soybean Aux/IAA genes. The normalized RNA-seq expression data of 19 tissues was used
to construct heatmap. Three samples from soybean seed compartments: GloEP (globular stage embryo proper), EmSCP (early maturation seed coat parenchyma),
and GloS (globular stage suspensor); 10 other tissues samples: Gs (globular stage seed), Hs (heart stage seed), Cs (cotyledon stage seed), Es (early maturation
stage seed), Ds (dry Seed), R (root), ST (stem), L (trifoliate leave), FB (floral bud), and WS (whole seedling 6 days after imbibition); three cotyledon development
samples: CoM (mid-maturation cotyledon), CoL (late maturation cotyledon), and CoS (seedling cotyledon); three early maturation seed parts: EcoEm (early
maturation embryonic cotyledon), EmEA (early maturation embryonic axis), and EmSC (early maturation seed coat). The lines show the syntenic blocks containing
the corresponding GmIAA genes, which experienced the WGD events. Gene names in red show dispersed duplicates. Color key at the bottom represents row wise
Z-score.
Frontiers in Plant Science | www.frontiersin.org 10 October 2015 | Volume 6 | Article 918

Singh and Jain Aux/IAA gene family in chickpea and soybean
embryos (Ploense et al., 2009). Another, gain-of-function mutant,
iaa12/bdl, also showed cotyledonary defects (Hamann et al.,
1999). Most of GmIAA genes showed relative low expression
levels in soybean CoS (seedling cotyledon), L (trifoliate leave), Hs
(heart stage seed), Es (early maturation stage seed), EcoEM (early
maturation embryonic cotyledon), and CoM (mid-maturation
cotyledon) tissues (Figure 5, Supplementary Table S7).
On the whole, the tissue-preferential expression exhibited by
several Aux/IAA genes in chickpea and soybean is indicative
of their involvement in biology of specific plant tissues and
developmental processes. It would be interesting to further
validate their functions in transgenics.
Overlapping and Differential Expression
Patterns of Duplicated GmIAA Genes
Duplicated genes possibly lead to subfunctionalization
(separation of original function), neofunctionalization (gain
of novel function), or nonfunctionalization (loss of original
function) based on their evolutionary fates (Prince and Pickett,
2002). Therefore, we also examined the functional redundancy of
duplicated GmIAA genes. In soybean, 50% of the paralogs from
the recent WGD event were found to be differentially expressed
and thus might have undergone functional divergence. Among
GmIAAs, 16 paralog pairs (GmIAA1/30, GmIAA3/28, GmIAA6/
33, GmIAA8/32, GmIAA9/48, GmIAA411/56, GmIAA13/58,
GmIAA17/24, GmIAA34/41, GmIAA36/63, GmIAA37/62, GmI-
AA38/61, GmIAA40/53, GmIAA43/54,GmIAA44/52, and
GmIAA45/51) representing segmental duplications shared
almost indistinguishable expression patterns (Figure 5,
Supplementary Table S7). On the contrary, the expression
patterns of another eight paralogs (GmIAA10/55, GmIAA14/59,
GmIAA15/18, GmIAA16/19, GmIAA21/26, GmIAA22/27,
GmIAA39/60, and GmIAA46/50) diversified significantly
(Figure 5). Interestingly, paralogs from much earlier duplication
events (legume WGD and gamma WGT) have more diverged
expression patterns. For example, three paralog gene pairs of
B2 subfamily, GmIAA5/35, GmIAA13/58, and GmIAA40/53
diverged into two clades after gamma WGT event. After
experiencing WGT, GmIAA40/53 formed one paralog pair, and
other was composed of GmIAA5/35 and GmIAA13/58 (Figure 5).
The former paralog gene pair was expressed during the stages of
seed development, but the two latter paralog gene pairs detached
after the legume WGD event, were highly expressed in the
GloEP (Figure 5, Supplementary Table S7). Other paralog genes
from different divergence events also showed similar expression
divergence (Figure 5). Besides gamma WGT, legume and Glycine
WGD also contributed to the expression divergence of paralog
GmIAA genes. For instance, paralog genes, GmIAA6/33 exhibited
higher expression in stem and FB, whereas GmIAA11/56 revealed
higher expression only in stem (Figure 5, Supplementary Table
S7). Similarly, paralog genes GmIAA22/27, separated from
Glycine WGD showed distinctively higher expression in Cs
FIGURE 6 | Expression profiles of CaIAA and GmIAA genes under abiotic stress conditions. (A) Heatmap shows differential expression of CaIAA genes
based on RNA-seq data. (B) qRT–PCR analysis of CaIAA genes under various stress treatments. Root and shoot control (CTR) was taken as a reference to
determine relative mRNA level under stress conditions. Error bars indicate standard error of mean. Data points marked with asterisk (∗P≤0.05, ∗∗P≤0.01, and
∗∗∗P≤0.001) indicate statistically significant difference between control and stress treatments. (C,D) Differential expression of GmIAA genes in response to drought
and salinity stress conditions. Color scale shows log2fold change relative to control sample. DS (desiccation), SS (salinity), CS (cold stress), V6 (vegetative stage
leaves), R2 (reproductive stage leaves).
Frontiers in Plant Science | www.frontiersin.org 11 October 2015 | Volume 6 | Article 918

Singh and Jain Aux/IAA gene family in chickpea and soybean
(cotyledon stage seed) and CoS (seedling cotyledon), respectively
(Figure 5, Supplementary Table S7). These results indicate
that gamma, legume and Glycine WGT events contributed
significantly in functional diversity of GmIAA gene paralogs.
Altogether, we can speculate that GmIAAs have been retained
by significant subfunctionalization in soybean during the course
of evolution. Meanwhile, it is interesting to note that most
of the paralog genes with similar expression profiles belong
to the same subfamily and grouped as sister pairs in the
phylogenetic tree (Figures 1 and 5). For example, two paralogs,
GmIAA13/58 and GmIAA5/35 in the same subfamily formed
sister pairs and displayed similar expression patterns (Figure 5).
The similar expression pattern of genes from same subfamily
of phylogenetic tree indicates that most of these genes may
have evolved coordinately in coding and regulatory (promoter)
regions, leading to their functional redundancy. Such functional
redundancy has been reported in Aux/IAA family in Arabidopsis
too (Overvoorde et al., 2005).
Differential Expression Patterns of
Aux/IAA Genes under Abiotic Stress
Plants are frequently exposed to environmental stresses, like
desiccation, salinity, and cold during their life cycle, which affect
their growth and development. Several reports highlighted that
the auxin-responsive genes were also engaged in various stress
responses (Ghanashyam and Jain, 2009;Jain and Khurana, 2009;
Wang et al., 2010a;Kumar et al., 2012;Cakir et al., 2013). To gain
more insights into the role of chickpea and soybean Aux/IAA
genes in abiotic stress tolerance, we analyzed their expression
profiles under desiccation, salinity, and cold stresses using RNA-
seq data for chickpea (Garg et al., 2015) and microarray data
for soybean. Many of the chickpea and soybean Aux/IAA genes
showed induction under desiccation, salinity and/or cold stresses
(Figure 6). For instance, transcript level of CaIAA3 was induced
significantly under desiccation in root, whereas it was induced in
both root and shoot under cold (Figures 6A,B, Supplementary
Table S8) and its promoter sequence harbors desiccation and
cold responsive cis-regulatory element (S000407; Supplementary
Table S6), indicating its role in desiccation and cold stress.
CaIAA7 showed induction under salinity in root and in shoot
under cold stress, while CaIAA13 was up-regulated in root under
salinity (Figures 6A,B, Supplementary Table S8). Transcript level
of CaIAA17 was found to be markedly induced in root under
desiccation and cold stresses (Figures 6A,B, Supplementary Table
S8), indicating its role in desiccation and cold stress responses.
However, CaIAA19 illustrated enhanced expression in root under
both desiccation and salt stresses (Figures 6A,B, Supplementary
Table S8), signifying its role in root under abiotic stresses. In
rice, OsIAA9 and OsIAA20 have been found to be induced
under both desiccation and salinity stress conditions (Jain and
Khurana, 2009). Further, putative salt stress-related cis-element
(S000453) was found in promoters of CaIAA3,7,13, and 19
(Supplementary Table S6), which has been demonstrated to be
responsible for salt stress response (Park et al., 2004). In response
to desiccation and salt stresses, the transcript level of CaIAA8
was suppressed in shoot and root (Figures 6A,B, Supplementary
Table S8), respectively, indicating that the function of this gene
is related to desiccation and salt stresses. Many SbIAA genes of
Sorghum bicolor have been found down-regulated under drought
conditions (Wang et al., 2010a). All the differentially expressed
CaIAAs were analyzed through qRT-PCR also and expression
patterns obtained from qRT-PCR and RNA-seq were correlated
well (Figures 6A,B).
In soybean, GmIAA57 revealed distinctly higher transcript
accumulation in vegetative stage (V6) leaves under drought
stress, while paralogous pair GmIAA47 and 49 showed noticeably
increased accumulation of transcripts in reproductive stage (R2)
leaves under drought stress (Figure 6C, Supplementary
Table S8). Their promoter sequences showed presence of
desiccation responsive cis-regulatory elements (S000174,
S000413; Supplementary Table S6), indicating their function in
drought stress responses. In response to salt stress, the transcript
levels of GmIAA4,5,8,27,46,54, and 55 were decreased in
seedling (Figure 6D, Supplementary Table S8), demonstrating
the function of these genes related to salt stress. Although the salt
stress-related cis-element (S000453) was found in the promoters
of GmIAA4,5,8,27,46,54, and 55 (Supplementary Table S4),
which is reported to induce the transcript level under salt stress
(Park et al., 2004), their expression levels were significantly down-
regulated under salt stress (Figure 6D, Supplementary Table
S8). This might indicate that some unidentified cis-regulated
elements may play an important role in regulating the expression
of these GmIAAs during stress responses in soybean. Moreover,
consistent with our result, many OsIAA genes (OsIAA7,8,12,14,
17,21,25, and 31) have also been reported to be suppressed in
rice under salt stress (Song et al., 2009). The present study clearly
revealed that the many of the Aux/IAA genes from chickpea
and soybean were expressed at significantly higher levels under
drought, cold, and salt treatments. It will be interesting to further
investigate them to understand their role in abiotic stresses
response/signaling.
CONCLUSION
In this study, we have performed a comprehensive analysis of
Aux/IAA genes in chickpea and soybean and provided insights on
the evolution of this gene family. The comprehensive expression
profiling indicated that members of Aux/IAA gene family are
involved in many plant responses during development and
abiotic stress conditions. Particularly, CaIAA1,3,4,11,12,13,15,
17,18, and 21 in chickpea and GmIAA6,13,22,23,31,33,35,39,
42,43,45,51,54,56,60, and 62 in soybean were found to have role
in various aspect of development, including root, stem, flower
bud, flower, and seed development. Further, CaIAA3,7,8,13, and
17 in chickpea and GmIAA4,5,8,27,46,47,54, and 55 in soybean
revealed their putative function in abiotic stress responses.
The presence of important cis-regulatory elements related to
various development processes and abiotic stress responses in
the promoter of these genes also provided insights into their
putative function. These genes are important candidates for
further functional characterization. Our analysis suggested that
the duplicated Aux/IAA genes may perform specific function due
to their subfunctionalization. Overall, information reported here
Frontiers in Plant Science | www.frontiersin.org 12 October 2015 | Volume 6 | Article 918

Singh and Jain Aux/IAA gene family in chickpea and soybean
for the CaIAAs and GmIAAs genes should facilitate further
investigations related to their functions in plant development and
stress responses.
FUNDING
This work was financially supported by the Department of
Biotechnology, Government of India, New Delhi, under the
Challenge Programme on Chickpea Functional Genomics.
ACKNOWLEDGMENTS
VS acknowledges the award of Senior Research Fellowship from
the Department of Biotechnology, Government of India.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online
at: http://journal.frontiersin.org/article/10.3389/fpls.2015.00918
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Conflict of Interest Statement: The authors declare that the research was
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