Báo cáo khoa học: Expression profile of PIN, AUX ⁄ LAX and PGP auxin transporter gene families in Sorghum bicolor under phytohormone and abiotic stress pot

Expression profile of PIN, AUX
⁄
LAX and PGP auxin
transporter gene families in Sorghum bicolor under
phytohormone and abiotic stress
ChenJia Shen
1
, YouHuang Bai
2,3
, SuiKang Wang
1
, SaiNa Zhang
1
, YunRong Wu
1
,
Ming Chen
1,2,3
, DeAn Jiang
1
and YanHua Qi
1
1 State Key Laboratory of Plant Physiology and Biochemistry, Zhejiang University, Hangzhou, China
2 Department of Bioinformatics, Zhejiang University, Hangzhou, China
3 James D. Watson Institute of Genome Sciences, Zhejiang University, Hangzhou, China
Introduction
Auxin plays a critical role in the spatiotemporal coor-
dination of plant growth and development, through
polar auxin transport [1–5]. Auxin transport proteins
in Arabidopsis are grouped into three families: auxin
resistant 1 ⁄ like aux1 (AUX1 ⁄ LAX) influx carriers,

pin-formed (PIN) efflux carriers and P-glycoprotein
Keywords
abiotic stresses; AUX ⁄ LAX; PGP; PIN;
Sorghum bicolor
Correspondence
Y. H. Qi, State Key Laboratory of Plant
Physiology and Biochemistry, Zhejiang
University, Hangzhou 310058, China
Fax: +86 571 88206133
Tel: +86 571 88981355
E-mail:
D. A. Jiang, State Key Laboratory of Plant
Physiology and Biochemistry, Zhejiang
University, Hangzhou 310058, China
Fax: +86 571 88206461
Tel: +86 571 88206461
E-mail:
Note
Proteins are shown in uppercase, genes are
shown in uppercase italics and mutants are
shown in lowercase italics
(Received 11 February 2010, revised 9 April
2010, accepted 10 May 2010)
doi:10.1111/j.1742-4658.2010.07706.x
Auxin is transported by the influx carriers auxin resistant 1 ⁄ like aux1
(AUX ⁄ LAX), and the efflux carriers pin-formed (PIN) and P-glycoprotein
(PGP), which play a major role in polar auxin transport. Several auxin
transporter genes have been characterized in dicotyledonous Arabidopsis,
but most are unknown in monocotyledons, especially in sorghum. Here, we
analyze the chromosome distribution, gene duplication and intron ⁄ exon

of SbPIN, SbLAX and SbPGP gene families, and examine their phylogenic
relationships in Arabidopsis, rice and sorghum. Real-time PCR analysis
demonstrated that most of these genes were differently expressed in the
organs of sorghum. SbPIN3 and SbPIN9 were highly expressed in flowers,
SbLAX2 and SbPGP17 were mainly expressed in stems, and SbPGP7 was
strongly expressed in roots. This suggests that individual genes might par-
ticipate in specific organ development. The expression profiles of these gene
families were analyzed after treatment with: (a) the phytohormones indole-
3-acetic acid and brassinosteroid; (b) the polar auxin transport inhibitors 1-
naphthoxyacetic acids, 1-naphthylphthalamic acid and 2,3,5-triiodobenzoic
acid; and (c) abscissic acid and the abiotic stresses of high salinity and
drought. Most of the auxin transporter genes were strongly induced by
indole-3-acetic acid and brassinosteroid, providing new evidence for the
synergism of these phytohormones. Interestingly, most genes showed simi-
lar trends in expression under polar auxin transport inhibitors and each
also responded to abscissic acid, salt and drought. This study provides new
insights into the auxin transporters of sorghum.
Abbreviations
ABA, abscissic acid; ABC, ATP-binding cassette; AUX1 ⁄ LAX, auxin resistant 1 ⁄ like aux1; BR, brassinosteroid; HMM, hidden Markov model;
IAA, indole-3-acetic acid; 1-NOA, 1-naphthoxyacetic acid; NPA, 1-naphthylphthalamic acid; PATI, polar auxin transport inhibitor; PGP,
P-glycoprotein; PIN, pin-formed; TIBA, 2,3,5-triiodobenzoic acid.
2954 FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS
(MDR ⁄ PGP ⁄ ABCB) efflux ⁄ conditional transporters
[6]. The PIN gene family was first cloned as an auxin
transporter from Arabidopsis [7], and has been
predicted in Brassica juncea, Cucumis sativus, Gossy-
pium hirsutum, Physcomitrella patens, Pisum sativum,
Populus tomentosa and Malus·domestica [8,9]. Many
PIN genes in dicotyledonous Arabidopsis have been
studied in detail, including AtPIN1–AtPIN4 and

AtPIN7, which act in auxin efflux transport, but the
function of AtPIN5, AtPIN 6 and AtPIN8 remains
unknown [4,5,10–14]. Reports on PIN genes in mono-
cotyledons are rare, although ZmPIN1a and ZmPIN1b
from maize may have a fundamental role in meristem
function, and point to a role for internal tissues in
organ positioning [15]. ZmPIN1-mediated auxin trans-
port is involved in cellular differentiation during maize
embryogenesis and endosperm development [16]. An
OsPIN1 gene expressed in the vascular tissues and root
primordia of rice was cloned and found to function in
auxin-dependent adventitious root emergence and til-
lering [17]. Recently, the expression pattern of the PIN
gene family has been comprehensively analyzed in rice,
and 12 OsPINs, including three monocot-specific PINs
(OsPIN9, OsPIN10a and OsPIN10b), were identified
using phylogenetic trees. OsPIN9 is highly expressed in
adventitious root primordia and pericycle cells on the
stem-base, suggesting that the monocot-specific PIN
protein may be involved in adventitious root develop-
ment [18].
The auxin influx carrier gene AUX1 encodes a
plasma membrane protein that belongs to the amino
acid permease family of proton-driven transporters,
and functions in the uptake of the Trp-like auxin mol-
ecule indole-3-acetic acid (IAA) [19–21]. The agravi-
tropic phenotype of aux1 mutant can be phenocopied
in wild-type seedlings using the auxin influx carrier
inhibitor 1-naphthoxyaceticacids (1-NOA), and rescued
using the membrane-permeable auxin 1-naphthylphtha-

lamic acid (1-NPA) [22–25]. AUX1 uses a novel traf-
ficking pathway in plants that is distinct from PIN
trafficking, and provides an additional mechanism for
the fine regulation of auxin transport [26]. The para-
logs of AUX1, LAX1, LAX2 and LAX3 maintain
phyllotactic patterning, and buffer the PIN-mediated
patterning mechanism against environmental or devel-
opmental influences [27]. The auxin influx carrier gene
LAX3 is induced by auxin, and increased LAX3 activ-
ity reinforces the auxin-dependent induction of a selec-
tion of cell-wall-remodeling enzymes, which are likely
to promote cell separation in advance of developing
lateral root primordia [28]. PaLAX1 from the wild
cherry Prunus avium, promotes the uptake of auxin
into cells and affects the content and distribution of
free endogenous auxin [29]. The AUX1 ⁄ LAX family of
auxin influx carriers is required for the establishment
of embryonic root cell organization in Arabidopsis tha-
liana [30]. In addition, AUX1 and LAX3 are involved
in auxin–ethylene interactions during apical hook
development in Arabidopsis seedlings [31].
P-Glycoprotein (PGP) proteins mediate the cellular
and long-distance transport of the phytohormone
auxin, and belong to a subfamily of the ATP-depen-
dent ATP-binding cassette (ABC) transporters.
AtPGP1 and AtPGP19 catalyze auxin export, whereas
AtPGP4 functions in auxin import [32]. Both pgp1 and
pgp19 from Arabidopsis
reduce growth and auxin
transport, and similar phenotypes are seen for pgp1

mutants of maize and sorghum, implying that PGP
functions as an auxin transporter [33–35]. Alterna-
tively, ABCB ⁄ PGP genes might respond to abiotic
factors in developmental regulation, because
PGP1 ⁄ ABCB1 regulates hypocotyl cell elongation in
light [36], the ABC transporter AtABCB14 is a malate
importer that modulates the stomatal response to CO
2
[37] and PGP19 expression is suppressed by the activa-
tion of phytochromes or cryptochromes [38].
The PGP, PIN and AUX ⁄ LAX families indepen-
dently transport auxin in both plants and heterologous
systems. However, PIN–PGP and AUX–PGP interac-
tions also function both independently and coordi-
nately to control polar auxin transport and impart
transport specificity and directionality [39]. PGP1–
PIN1 or PGP19–PIN1 coexpression synergistically
increases IAA export, whereas coexpression of PGP1–
PIN2 and PGP19–PIN2 shows an antagonistic effect;
PGP4–PIN2 coexpression enhances auxin uptake,
whereas PGP4–PIN1 reverses this effect, suggesting
that specific PIN–PGP pairings regulate auxin trans-
port in specific tissues. Similarly, an antagonistic effect
is also observed in AUX1–PGP4 and AUX1–PGP1 co-
expression [4,39–41]. A newly developed Schizosaccharo-
myces pombe system of co-expression for studying the
comparative and structural characterizations of plant
transport proteins would facilitate understanding of
the coordination between the PIN, AUX ⁄ LAX and
PGP gene families in auxin transport [41].

To understand auxin response and transport, we
analyzed the structural characteristics and expression
profiles of genes for auxin ⁄ indole-3-acetic acid, auxin
response factor, Gretchen Hagen 3, small auxin up
RNAs and lateral organ boundaries in sorghum under
abiotic stress, which is related to the auxin response
[42]. This article is a companion to this research,
predicts the members of the auxin transporter PIN,
AUX ⁄ LAX and PGP gene families, and analyzes their
chromosomal distribution, gene duplication and
C. Shen et al. Auxin transporter gene families in Sorghum bicolor
FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS 2955
phylogenic relationships. The organ-specific expression
and expression profiles of the three gene families in
sorghum under IAA, brassinosteroid (BR), polar auxin
transport inhibitors (PATIs) and abiotic stress control
were analyzed using real-time PCR.
Results and Discussion
Chromosomal distribution and gene duplication
The ancestor of monocots is assumed to have under-
gone whole genome duplication once,  70 million
years ago, before the divergence of rice, sorghum and
maize [43]. Whole genome duplication provided gene
families with the opportunity to grow during evolution
of the angiosperms and is always followed by gene loss,
which may explain why some gene pairs survive in
duplication and others do not [44]. Of the 40 genes in
this study, 26 were located in the duplication region by
chromosome mapping. The gene pairs in the duplica-
tion regions were SbPIN1–SbPIN8, SbPIN6–SbPIN10,

SbLAX3–SbLAX5, SbPGP5–SbPGP23, SbPGP6–
SbPGP24 and SbPGP14–SbPGP21 (Fig. 1). Of the 26
genes, the other 14 are retained, but represent only one
copy in the duplication region. Tandem duplication
was also important in the evolution of the SbPGP gene
family. We observed that four distinct tandem dupli-
cate gene clusters represented 10 SbPGP genes: two
clusters with two tandem genes (SbPGP19–20 and
SbPGP23–24), and another two clusters containing
three tandem genes (SbPGP5–7 and SbPGP10–12).
Analysis of gene structure
SbPIN
The PIN gene family is only found in land species [45].
Eleven PIN genes have been identified in sorghum
(Fig. S1 and Table S1). Similar to the AtPIN and
OsPIN proteins [14,18], the SbPIN proteins have a
highly conservative domain architecture, with two
hydrophobic domains divided by a hydrophilic loop of
three conserved regions, the C1–C3 domains, and two
separate variable regions, V1 and V2 [8]. The internali-
zational motif NPNXY [46] is found between the
hydrophilic loop and the C-terminal hydrophobic
domain of all SbPIN proteins except SbPIN4, in which
the first amino acid is an isoleucine rather than aspara-
gine [47]. SbPIN1, -3, -4, -5 and -8 have a short hydro-
philic loop lacking the V1 and V2 regions, whereas the
other SbPIN family members have the full-length
hydrophilic loop. Some sites in the central hydrophilic
loop can be phosphorylated by serine ⁄ threonine pro-
tein kinases such as PINOID kinase [45]. Most SbPINs

contain two possible phosphorylation sites: one is
marked by two asterisks (Fig. S1) because it is not
known which of the two adjacent amino acids is phos-
phorylated, and the other possible site is marked by
only one asterisk.
In all AtPIN proteins, the hydrophobic domains are
suggested to contain five transmembrane helices [45].
According to the TMHMM server prediction (http://
www.cbs.dtu.dk/services/TMHMM), the hydrophobic
domain contains multiple transmembrane helices. In
the C-terminus of the SbPIN5 protein, deletion of a
segmental sequence decreases the number of trans-
membrane helices.
SbLAX
The length of the five SbLAX proteins ranges from
487 to 553 amino acids, and the core regions of LAX
proteins are highly conserved, with 10 transmembrane
helices predicted by bioinformatics for each member of
the SbLAX family (Fig. S2 and Table S1). In SbLAX
proteins, the N-terminus is rich in acidic amino acids
and the C-terminus is proline-rich.
SbPGP
The PGP family belongs to the ABCB subgroup of the
ABC transporter superfamily [48]. Multiple sequence
alignment showed that almost all SbPGP proteins
share a common domain architecture with two similar
modules [41]. A transmembrane domain and a nucleo-
tide-binding domain are connected by an intracellular
loop in the N- and C-termini of SbPGP proteins
(Fig. S3 and Table S1). Each transmembrane domain

is composed of six transmembrance helices, as pre-
dicted by the TMHHH webserver. In addition to two
conserved modules, a less-conserved linker domain
connecting the first nucleotide-binding domain and the
second transmembrane domain is seen in all SbPGPs.
A second nucleotide-binding domain in the C-terminus
of SbPGP10 proteins is absent.
Exon–intron structure analysis
In addition to phylogenetic analysis, the exon–intron
structures of SbPIN, SbLAX and SbPGP genes were
examined (Fig. S4A–C). All the ‘long’ SbPINs have a
conserved intron phase pattern, whereas most of the
‘short’ PINs in sorghum do not, except for SbPIN3.
The intron phase pattern also can be detected in the
exon–intron organization of SbLAX. In the SbPGP
gene family, each group has a highly similar exon–
intron structure.
Auxin transporter gene families in Sorghum bicolor C. Shen et al.
2956 FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS
Analysis of cis-element in promoter in abiotic stress
Scanning for cis-acting regulatory DNA elements
within the promoters of SbPIN, SbLAX and SbPGP
genes (2.5 kb from the start codon) using an in-house
perl script, revealed that the promoters of the three
gene families contain numerous DNA elements pre-
dicted to respond to auxin, abscissic acid (ABA),
drought and high salt (Table S2). The DNA elements
include multiple copies of TGTCTC (AuxREs, auxin
response factor binding) [49], ACGTG (drought-
inducible, ABRE-like element) [50], CACGTG

(ABA-inducible) [51], and GNGGTG, GTGGNG and
GAAAAA (salt-inducible) [52,53]. The results of cis-
element analysis suggested that the functions of these
genes may be associated with environmental stress,
which prompted us to investigate the relationship
between these auxin transporter genes and abiotic
stress.
Phylogenetic relationship of PIN, LAX and PGP in
Arabidopsis, rice, and sorghum
To investigate the evolutionary relationship of the
three classes of proteins identified as auxin transporter
proteins, multiple sequence alignment of all full-length
proteins was conducted using the RAxML webserver
for phylogenetic analysis with the maximum likelihood
method. SbPIN proteins clustered into five groups in
the phylogenetic tree (Fig. 2A). According to the
length of the distinct central hydrophilic loop, the PIN
proteins were classified into two broad subfamilies
[45]: ‘short’ PINs (SbPIN1, -3, -5 and -8) and ‘long’
PINs (SbPIN2, -4, -6, -7 and -9–11). Phylogenetic anal-
ysis indicated that the ‘long’ PINs form groups 2–5,
and group 1 is comprised of the ‘short’ PINs: two
AtPINs, four OsPINs and four SbPINs. Group 3 con-
tains only one AtPIN protein (AtPIN1) compared with
the three members (SbPIN6, -7 and -10) in sorghum,
Fig. 1. Chromosome mapping of SbPIN, SbLAX and SbPGP gene families. The genome visualization tool CIRCOS was employed. Sorghum
chromosomes are arranged in a circle, and the centromere of each chromosome is marked in black. Ribbon links represent the segmental
duplication region retrieved from the SyMAP database [72]. SbPIN, SbLAX and SbPGP genes are mapped by locus.
C. Shen et al. Auxin transporter gene families in Sorghum bicolor
FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS 2957

AB
C
Fig. 2. Phylogenetic tree of the PIN, LAX and PGP families in Arabidopsis, rice and sorghum. Gene families names in are black for Arabidop-
sis, red for rice and blue for sorghum. Bootstrap values are presented for all branches. (A) PIN: data on AtPIN and OsPIN families (Tables S3
and S4) is based on TAIR annotation and Wang et al. [18]. (B) LAX: Inventory of the AtLAX and OsLAX family is based on TAIR and
TIGR rice databases (Tables S3 and S4). (C) PGP: inventory of AtPGP and OsPGP family is based on the ABC superfamily review by Verrier
et al. [48].
Auxin transporter gene families in Sorghum bicolor C. Shen et al.
2958 FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS
and four (OsPIN1a–d) in rice (Fig. 2A and Tables S3
and S4). This result, combined with the analysis of the
OsPIN family in rice, indicated that the growth of the
PIN family in monocots can be attributed to whole
genome duplication in the monocot ancestor, after the
divergence between dicots and monocots.
The AUX ⁄ LAX sequences among plant species are
highly similar [23]. Sorghum and rice have five mem-
bers, one more than Arabidopsis (Fig. 2B, and
Tables S3 and S4). The phylogenetic tree for LAX is
consistent with two major clades: one clade contains
AtAUX1, AtLAX1 and two members in sorghum and
rice (SbLAX1, -2, -4 and OsLAX2, -4, -5) and the
other clade contains AtLAX2 and AtLAX3, and three
members of SbLAX and OsLAX family.
Phylogenetic analysis of PGPs in the three genomes
indicated that the PGP family can be divided into
three groups [30]. AtPGP1, AtPGP4 and AtPGP19 are
well characterized in Arabidopsis and can be placed
into groups 1 and 2 (Fig. 2C, and Tables S3 and S4).
SbPGP21 is close to AtPGP1, and two SbPGPs

(SbPGP16 and SbPGP18) are close to AtPGP19 in
group 1. In group 2, the AtPGP4 clusters with other
AtPGPs, but without any PGP in rice and sorghum.
All genes in the SbPIN, SbLAX and SbPGP families
were apparently closer to rice than Arabidopsis, by phy-
logenetic trees (Fig. 2A–C), and most genes, with one
rice gene, formed individual sister pairs (1 : 1 ortholo-
gous relationships). However, in group 3, a gene cluster
was comprised of four SbPGPs (SbPGP10–13) and the
OsPGP9 gene (n : 1 orthologous relationship). In par-
ticular, the SbPGP10–12 and OsPGP9 gene pair was
one of four tandem gene pairs in the sorghum genome.
Each member of the three tandem gene groups
(SbPGP5–7, SbPGP19–20 and SbPGP23–24) formed a
sister pair with one OsPGP gene, and the rice genes
could also be grouped into three tandem gene pairs on
rice chromosomes (Fig. 2C). Thus, the SbPGP family
in sorghum has undergone tandem duplication at two
different times. Before the divergence of sorghum and
rice, the ancestors of the three gene pairs (SbPGP5–7,
SbPGP19–20 and SbPGP23–24) all arose through
tandem duplication events to create the gene pairs of
sorghum and rice. Tandem duplication must have
happened in sorghum only after divergence from rice
 70 million years ago, accounting for the n : 1 orthol-
ogous relationship between SbPGP10–12 and OsPGP9.
Organ-specific expression of SbPIN, SbLAX and
SbPGP genes in sorghum
To determine the expression level of each SbPIN,
SbLAX and SbPGP gene in different organs, real-time

PCR was performed with total RNA from sorghum
leaf, stem, root and flower. Real-time RT–PCR analy-
sis showed that expression of most SbPINs was con-
stitutive in all tissues, consistent with results from rice
[18]. However, SbPIN3 and SbPIN9 were more highly
expressed in flowers than in other organs (Fig. 3).
OsPIN5b is expressed in young panicle [18], and
atpin1
mutants exhibit pinformed inflorescences and reduced
basipetal auxin transport in inflorescence axes
[7,51,54], whereas ZmPIN1b increases during female
inflorescence development [15]. These results implied
that the PIN genes were related to the growth and
development of flower organs. The ataux1 mutant phe-
notype is complemented by strong expression of
PaLAX1 (Prunus avium), causing multiple inflorescenc-
es [29], and suggesting that the function of LAX1 is in
inflorescence development. In sorghum, the SbLAX
genes were differently expressed in each organ, except
for SbLAX2, which was highly expressed in stems. Sim-
ilar to AtPGP1 [55], most of the SbPGP genes did not
exhibit a tissue-specific expression pattern, although
SbPGP17 was expressed in stem, which showed its
transcription was organ specific. SbPGP7 (Fig. 3) and
AtPGP4 [56] were strongly expressed in roots, and
weakly expressed in stems, leaves and flowers, suggest-
ing that SbPGP is like AtPGP, which functions in a tis-
sue-specific manner, similar to the PIN proteins [56,57].
In addition, SbPIN4 and -5, SbLAX3 and SbPGP2,-3,
-5,-9,-10,-13,-15,-20,-23 and -24 showed almost no

expression under normal growth conditions, and their
relative expression level was < 0.5 compared with
SbACTIN expression, defined as 1000.
Most SbPIN, SbLAX and SbPGP genes are
induced by IAA and BR
The action of plant hormones in regulating physiology
and development often involves extensive cross-talk
between different signaling pathways [58]. Auxin and
BR exert similar physiological effects through synergis-
tic interaction [59]. Many auxin response genes are
also regulated by BR [60–63]. To determine if auxin
transporters are also involved in phytohormone signal-
ing, we obtained expression profiles for the three auxin
transporter gene families, SbPIN, SbLAX and SbPGP.
Nearly all SbPIN genes were upregulated by IAA
treatment, except for SbPIN1 and SbPIN5, which were
downregulated in leaf ⁄ root, and SbPIN4
which was
downregulated in root (Fig. 4A,C). Compared with
leaves, SbPIN genes in roots responded rapidly to IAA
and BR, especially SbPIN8 and -9. Specifically, all
genes except SbPIN3 were upregulated in roots by BR
treatment, with less dramatic changes in leaves. In rice,
C. Shen et al. Auxin transporter gene families in Sorghum bicolor
FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS 2959
SbPGP22
0
1
2
3

4
5
6
SbPIN1
0
5
10
15
20
SbPIN2
0
5
10
15
20
SbPIN4
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
SbPIN5
0
0.1
0.2
0.3
0.4

SbPIN6
0
1000
2000
3000
4000
5000
6000
SbPIN7
0
1
2
3
4
5
6
7
SbPIN8
0
0.5
1
1.5
2
2.5
SbPIN9
0
5
10
15
20

SbPIN10
0
50
100
150
200
SbLAX1
0
5
10
15
20
25
30
35
SbLAX2
0
2
4
6
8
10
12
SbLAX3
0
0.05
0.1
0.15
0.2
SbLAX4

0
0.5
1
1.5
2
2.5
3
SbLAX5
0
20
40
60
80
100
SbPGP1
0
0.5
1
1.5
2
2.5
SbPGP2
0
0.01
0.02
0.03
0.04
0.05
SbPGP3
0

0.1
0.2
0.3
0.4
0.5
0.6
SbPGP4
0
0.2
0.4
0.6
0.8
1
SbPGP5
0
0.01
0.02
0.03
0.04
0.05
SbPGP8
0
5
10
15
20
25
30
SbPGP9
0

0.02
0.04
0.06
0.08
0.1
SbPGP10
0
0.05
0.1
0.15
0.2
0.25
0.3
SbPGP11
0
20
40
60
80
100
120
SbPGP12
0
1
2
3
4
5
SbPGP13
0

0.02
0.04
0.06
0.08
0.1
0.12
0.14
SbPGP14
0
1
2
3
4
SbPGP15
0
0.05
0.1
0.15
0.2
SbPGP16
0
2
4
6
8
10
12
SbPGP17
0
0.5

1
1.5
2
2.5
3
SbPGP18
0
0.2
0.4
0.6
0.8
1
1.2
SbPGP19
0
2
4
6
8
10
12
14
SbPGP20
0
0.1
0.2
0.3
0.4
SbPGP21
0

0.5
1
1.5
2
2.5
3
3.5
SbPGP23
0
0.005
0.01
0.015
0.02
0.025
0.03
SbPGP24
0
0.05
0.1
0.15
0.2
0.25
0.3
SbPIN11
0
10
20
30
40
50

RNA relative level
SbPGP6
0
10
20
30
40
50
60
70
SbPIN3
0
100
200
300
400
500
SbPGP7
0
10
20
30
40
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR LFSR

LFSR
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR
LF SR
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR
LFSR

LFSR
LFSR
Fig. 3. Analysis of tissue-specific expression of SbPIN, SbLAX and SbPGP genes. Real-time quantitative RT-PCR of SbPIN, SbLAX and
SbPGP genes. Total RNA was extracted after 3 weeks, from leaves, stems and roots. Young panicles of sorghum were planted in Murashi-
ge and Skoog nutritional liquid medium. Relative mRNA levels of individual genes normalized to SbACTIN (Sb01g010030.1) gene are shown.
The abscissa shows the relative RNA expression level; the ordinate shows different tissues. L, leaf; F, flower; S, stem; R, root. Samples
were analyzed as independent biological replicates from three different RNA isolations, and cDNA syntheses and error bars are for cDNAs
measured in triplicate.
Auxin transporter gene families in Sorghum bicolor C. Shen et al.
2960 FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS
most OsPIN genes were induced by IAA, except for
OsPIN2 and OsPIN9. OsPIN1c was also induced by
BR, whereas OsPIN5a was repressed [18].
All LAX genes were upregulated in Arabidopsis root
by IAA treatment [14]. In sorghum, SbLAX2 and
SbLAX3 were induced by IAA (Fig. 4B), but expres-
sion levels of SbLAX1 and SbLAX4 were completely
inhibited in leaf and root, and SbLAX5 was inhibited
in leaf. By contrast, all five SbLAX genes were upregu-
lated by BR treatment in root (Fig. 4D). SbLAX1 and
SbLAX4 were downregulated by BR in leaf.
Under IAA treatment, several SbPGP genes, includ-
ing SbPGP1,-13,-15,-18 and -23, were upregulated in
root. AtPGP1 expression is auxin-responsive, and the
PGP1 promoter contains ARE motifs for ARFAT,
ASF-1 and NtBBF1 [40]. SbPGP5,-11,-12,-17,-19
and -24 were inhibited in both leaf and root by IAA
treatment (Fig. 4E,F). BR treatment upregulated
SbPGP2 and SbPGP5 in leaves and roots, but
SbPGP4 and -18 were upregulated only in roots.

SbPGP3,-6,-10,-16,-17,-20,-21 and -22 were inhib-
ited by BR treatment.
Many auxin influx ⁄ efflux carriers are induced by
exogenous auxin, suggesting that changes in auxin
concentration are mediated at both the tissue and cel-
lular levels [14]. This is supported by our results. In
addition, although a close relationship between auxin
and BR has been widely reported, the molecular
mechanism for combinatorial control of shared target
genes has remained elusive [63]. Recent studies and
the data presented here provide experimental evidence
for the synergistic effect of IAA and BR in the plant
response to hormone signaling. For example,
OsPIN5a was downregulated by auxin and BR [18],
expression of PGP4 in Arabidopsis increased under
IAA and BR [41], and SbPIN2,-6–10, SbLAX4,
SbLAX5, SbPGP6–11,-13,-17-19 and -21–24 in
leaves and roots showed the same expression trend
under both IAA and BR treatments (Fig. 4E,F).
Furthermore, this effect has been observed phenotypi-
cally, for example, in the synergistic promotion of
lateral root development by auxin and BR which
increases acropetal auxin transport in Arabidopsis
[59].
0
5
10
15
20
25

30
35
SbPIN1
SbPIN2
SbPIN3
SbPIN4
Sb
PIN5
Sb
PIN6
Sb
PIN
7
Sb
PIN
8
Sb
PI
N9
S
bP
IN1
0
S
b
PIN1
1
0
2
4

6
8
10
SbLA
X
1
Sb
LAX
2
S
bLAX
3
SbLA
X
4
SbL
AX
5
0
1
2
3
4
5
6
S
b
P
GP
1

S
b
P
GP
2
SbPGP
3
SbPGP4
SbPGP5
SbPGP6
S
b
P
G
P7
S
b
P
GP
8
S
b
P
GP
9
S
b
P
GP
1

0
SbPG
P
1
1
SbPG
P
1
2
SbPG
P
1
3
SbPGP14
S
b
P
GP15
S
b
P
GP16
S
b
P
GP
1
7
SbPG
P

1
8
SbPG
P
1
9
SbPGP20
SbPGP21
S
b
P
GP22
S
b
P
GP
2
3
S
b
P
GP
2
4
RNA relative level
Leaf
Root
0
5
10

15
0
200
250
SbPIN
1
SbPIN
2
SbPIN
3
SbPIN
4
SbPIN
5
SbPIN
6
SbPIN
7
SbPIN
8
SbPIN
9
Sb
PIN10
S
bPIN11
0
5
10
15

20
Sb
LAX1
Sb
LAX2
Sb
LAX3
Sb
LA
X4
SbLAX5
0
5
10
15
20
25
S
bPGP1
S
b
P
G
P2
SbPGP3
SbPGP4
S
bPG
P5
S

b
P
G
P6
SbPGP7
S
bPG
P8
S
b
P
G
P9
SbPG
P1
0
SbPG
P1
1
SbPG
P
12
SbPG
P13
SbPG
P1
4
SbPG
P
15

SbPG
P16
SbPG
P1
7
S
bPG
P18
SbPG
P19
SbPG
P2
0
S
bPG
P21
SbPG
P22
SbPG
P23
S
bPG
P24
IAA treatment
tnemtaertAAItnemtaertAAI
BR treatment
BR treatment
BR treatment
AB
C

E
F
D
Fig. 4. Expression profiles of auxin trans-
porter SbPIN, SbLAX and SbPGP genes
under IAA and BR treatment. Total RNA
was extracted from 3-week-old seedlings
treated under indicated conditions. The rela-
tive RNA level of genes after treatment,
compared with the expression of genes in
leaves and roots planted in Murashige and
Skoog medium. (A–E) show expression of
SbPIN, SbLAX and SbPGP genes under IAA
and BR treatment. IAA treatment: 10 l
M
IAA for 3 h; BR treatment: 1 lM BR for
12 h. Real-time PCR conditions were as in
Fig. 3. (A), (B) and (E) show expression lev-
els of SbPIN, SbLAX and SbPGP genes
under IAA treatment. (C), (D) and (F) show
expression levels of SbPIN, SbLAX and
SbPGP genes under BR treatment.
C. Shen et al. Auxin transporter gene families in Sorghum bicolor
FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS 2961
Similar expression trends under PATIs treatment
It is known that 1-NOA, 1-naphthylphthalamic acid
(NPA) and 2,3,5-triiodobenzoic acid (TIBA) are PATIs.
NOA is an auxin influx carrier inhibitor, and TIBA and
NPA are auxin efflux carrier inhibitors, and are used to
facilitate studies on auxin influx ⁄ efflux carriers [23,61–

63] To gain insight into the influence of PATIs on auxin
transporters, we analyzed the transcriptional fluctuation
of the three gene families, SbPIN, SbLAX and SbPGP
under NOA, NPA and TIBA treatment. Surprisingly,
the three families showed no distinct differences in tran-
scriptional level after PATI treatment. For example,
SbPIN1–3,-5 and -11 were expressed similarly under
inhibitor treatment; SbPIN4 was highly upregulated in
both leaves and roots when treated with TIBA, but only
in leaves with 1-NOA and NPA treatment (Fig. 5A,C
and E). SbPIN8 and -9 were upregulated > 10-fold by
0
10
20
30
80
100
SbPIN1
SbPIN2
SbPIN3
SbPIN4
SbPIN5
SbPIN6
SbPIN7
SbPIN8
SbPIN9
Sb
PIN10
SbPIN11
0

10
20
30
SbPIN1
SbPIN2
SbPIN3
SbPIN4
SbPIN5
SbPIN6
SbPIN7
SbPIN8
SbPIN9
Sb
PIN10
SbPIN11
0
5
10
15
0
5
10
15
20
25
SbPGP
1
SbPGP2
SbPGP3
SbPGP4

SbPGP5
SbPGP6
SbPGP7
SbPGP8
SbPGP9
SbPGP10
Sb
P
GP11
Sb
PG
P12
Sb
P
GP13
Sb
PG
P14
Sb
PG
P15
Sb
PGP
16
Sb
PG
P17
Sb
PGP
18

Sb
PGP1
9
Sb
PGP2
0
Sb
PGP2
1
Sb
PGP2
2
Sb
PGP23
Sb
PGP24
RNA relative level
0
10
20
30
40
50
60
SbPI
N
1
SbPI
N
2

SbP
IN3
SbP
IN4
SbP
IN5
SbPIN6
SbPIN7
SbPI
N
8
SbPI
N
9
SbPI
N
10
SbPIN11
0
2
4
6
8
10
12
SbLAX1
SbLAX2
SbLAX3
SbLAX4
SbLAX5

SbLAX1
SbLAX2
SbLAX3
SbLAX4
SbLAX5
0
5
10
15
20
25
30
SbPIN1
S
b
PI
N
2
SbP
I
N3
S
b
PI
N
4
S
bP
I
N5

S
b
PIN6
S
bP
I
N
7
SbPIN8
S
b
PI
N
9
Sb
P
IN10
SbPIN11
0
5
10
15
20
25
30
35
SbLAX
1
SbL
A

X2
SbL
A
X3
SbLAX4
Sb
L
AX
5
0
5
10
15
20
25
30
35
Sb
P
GP
1
Sb
PG
P
2
SbPGP3
Sb
P
GP
4

Sb
PG
P
5
S
b
PGP6
SbPGP
7
Sb
P
GP
8
S
b
PG
P
9
Sb
PG
P1
0
SbP
G
P11
S
bPGP12
Sb
PG
P

13
SbP
G
P14
SbPGP15
S
bPG
P
16
SbP
G
P1
7
SbPGP18
S
bPGP19
Sb
PG
P2
0
SbP
G
P21
S
bPGP22
Sb
PG
P
23
SbP

G
P2
4
0
5
10
15
20
25
30
35
40
SbPGP1
SbPGP2
Sb
PG
P3
SbPGP4
SbPG
P
5
SbPGP6
SbPGP7
SbPGP8
SbPGP9
bPGP10S
bPGP11S
bPGP12S
b
S

P
GP13
bPGP14
S
bPGS
P1
5
bPGP16
S
bPGP17S
bPGP18
S
bPGP19
S
b
S
P
GP20
bPGP21
S
bPG
S
P2
2
bPGP23
S
bPGP24
S
Leaf
Root

1-NOA treatment
1-NOA treatment
1-NOA treatment
NPA treatment
NPA treatment
NPA treatment
TIBA treatment
TIBA treatment
TIBA treatment
A
B
C
E
F
G
H
I
D
Fig. 5. Expression profiles of auxin trans-
porter genes SbPIN, SbLAX and SbPGP
under auxin transport inhibitor treatment.
Seedlings (3 weeks old) were treated with
30 l
M 1-NOA, 25 lM NPA or 50 lM TIBA for
3 h. Real-time PCR conditions were as in
Fig. 3. (A), (B) and (G) show expression
levels of SbPIN, SbLAX and SbPGP genes
under NOA treatment. (C), (D) and (H) show
expression levels of SbPIN, SbLAX and
SbPGP genes under NPA treatment. (E), (F)

and (I) show expression levels of SbPIN,
SbLAX and SbPGP genes under TIBA
treatment.
Auxin transporter gene families in Sorghum bicolor C. Shen et al.
2962 FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS
all treatments. SbPIN1 and -7 were downregulated
by NOA; SbPIN2,-6 and -10 were downregulated by
NPA; SbPIN6 and -10 were downregulated by TIBA.
Most SbLAX genes were upregulated in roots by
PATIs, except for SbLAX3 which was upregulated in
leaves by NPA (Fig. 5B,D,F). Expression of SbLAX1,
-2,-4 and -5 was stable in leaves. SbPGP genes showed
similar responses to different PATIs, or displayed only
slight variations (Fig. 5G,H,I). For example, SbPGP2
and -14 were upregulated  20-fold by the PATIs, espe-
cially in roots. Many SbPGP genes were downregulat-
ed, including SbPGP6,-7,-8,-11,-12,-16 and -24.
SbPGP1,-3 and -15 were more sensitive to 1-NOA and
NPA. AtPGP1 expression was also NPA-sensitive and
NPA treatment reversed increases in PGP1 expression
[40]. Previous microarray data on auxin response genes
in Arabidopsis showed that TIBA has a stronger effect
than NPA when used at the same concentration, and
TIBA regulated a greater number of genes than NPA:
nine genes were upregulated and 19 downregulated
under NPA treatment, whereas 473 genes were upregu-
lated and 332 downregulated under TIBA treatment
[14]. However, we did not find an increased effect in
these auxin transporter genes, even with a TIBA con-
centration twice that of NPA. These results suggested

that transcription of auxin transporter genes was con-
trolled by the auxin transport inhibitors, but had no
direct connection to concentration, at least in the range
tested.
Function of auxin transporters might be related
to ABA and abiotic stress
Auxin primarily acts in many developmental processes,
and ABA mediates various abiotic and biotic stress
responses in plants. Recent studies suggest that auxin
is also involved in stress or defense responses, and a
significant number of auxin-responsive genes are impli-
cated in abiotic stress responses [64–67]. Various envi-
ronmental and endogenous signals modulate
trafficking and polarity of PIN proteins and change
auxin distribution by this mechanism [58]. To address
whether auxin transport genes are also involved in abi-
otic stress responses in sorghum, their expression pro-
file was analyzed using real-time PCR. Statistical
analysis showed that the expression of most genes was
up- or downregulated under ABA, salt and drought
treatments (Fig. 6). In particular, SbPIN1–6 and -9,
SbLAX1 and -3, and SbPGP4,-5,-9,-14
and -19–21
showed similar transcriptional fluctuation trends in
roots and leaves under the three stress treatments. The
expressions of SbPIN4,-5,-8,-9 and -11 were highly
increased, whereas SbPIN1–3,-6,-7 and -10 were
almost inhibited by all three treatments. The expres-
sion level of SbLAX1,-2,-4 and -5 compared with
SbLAX3 in leaves was lower than in roots when trea-

ted with ABA. However, the response of SbLAX genes
to salt and drought stresses was irregular, with
SbLAX4 expression downregulated dramatically under
the stresses (Fig. 6B,D,F). Interestingly, transcription
of the SbPGP gene family was almost inhibited in
roots under salt treatment (Fig. 6G–I). SbPGP1,-2,-5,
-13,-14 and -15 were induced in roots under ABA
treatment, whereas SbPGP2,-3,-4,-7,-12 and -23
were induced in leaves under salt or drought stress.
Under salt and drought treatment, SbPGP13,-15,-17,
-18,-20,-21 and -24 were all downregulated in both
leaves and roots. PGP genes respond to some abiotic
factors such as light, CO
2
, phytochromes and crypto-
chromes [36–38]. However, the PGP gene response to
ABA or salt and drought stress has rarely been
reported, although expression of PGP4 in Arabidopsis
is reduced with ABA treatment [56]. We first analyzed
the expression profile of PGP genes under ABA, salt
and drought treatment, and found that the expression
trends of many SbPGP genes (except for SbPGP2,-3,
-16,-22 and -23) under salt and drought treatment
were similar (Fig. 6G–I). This similarity was also seen
in the SbPIN and SbLAX genes (Fig. 6C–F), suggest-
ing that the function of the auxin transport genes
might also be involved in the abiotic stresses of salt
and drought, and respond to both stresses with similar
expression patterns. Salt stress has recently been
reported to promote auxin accumulation in developing

primordia, and stimulates a stress-induced morpho-
genic response in Arabidopsis roots [68]. Moreover, in
the auxin transporter mutant aux1–7, the lateral root
proliferation component of the salt stress-induced mor-
phogenic response is completely abrogated. This pro-
vides genetic and physiological evidence that the auxin
influx carrier is involved in the response to salt stress.
To understand the relationship between auxin trans-
porter and abiotic stress in detail, a combination of
molecular biology, reverse genetics and plant physiol-
ogy may help to identify the biological function of
each transporter. For example, loss- or gain-of-func-
tion mutants of auxin transporters can be obtained
through T-DNA insertion or activation–tagging meth-
ods to aid experiments in the regulation mechanisms
of auxin-abiotic stress signaling.
In conclusion, the comprehensive gene structure and
transcription analysis of the auxin transporter genes
SbPIN, SbLAX and SbPGP in sorghum, including
expression under abiotic stress, was reported here. The
expression level of these auxin transporters was
affected by IAA and BR, and most genes showed
C. Shen et al. Auxin transporter gene families in Sorghum bicolor
FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS 2963
similar expression trends under the PATIs, NOA,
TIBA and NPA. In addition, gene family members
also responded to ABA, salt and drought. This study
presented useful bioinformational data for the auxin
transporters of sorghum, and provided evidence for
the role of auxin transporters in mediating cross-talk

between the auxin response and various abiotic stres-
ses. The action of auxin transporters under cross-talk
regulation will be an important subject for detailed
future studies.
0
10
20
30
40
50
60
S
bP
IN
1
S
bP
IN
2
S
bP
IN
3
S
bPI
N
4
S
bPI
N

5
S
bPI
N
6
S
bPI
N
7
S
bPI
N
8
S
bPIN9
S
bPIN10
S
bPIN11
S
0
0.5
1
1.5
2
2.5
3
3.5
4
b

L
A
X1
S
b
L
A
X2
S
b
L
A
X3
S
b
LAX4
S
b
LAX5
0
5
10
15
20
25
SbPGP
1
SbPGP2
SbPGP3
SbPGP4

SbPGP5
SbPGP6
SbPGP7
SbPGP8
SbPGP9
Sb
PGP10
Sb
PGP11
Sb
PGP12
Sb
PGP13
Sb
PGP14
Sb
PGP15
Sb
PGP16
S
b
PGP17
S
b
PGP18
Sb
P
GP19
Sb
PG

P20
Sb
PGP
21
Sb
PGP
22
Sb
PGP23
Sb
PGP24
RNA relative level
0
1
2
3
4
5
6
7
8
9
Sb
P
IN1
Sb
PI
N2
S
b

PI
N3
S
b
PI
N4
SbPIN5
SbPIN6
Sb
P
IN7
Sb
PI
N8
S
b
PI
N9
Sb
P
IN1
0
Sb
PI
N1
1
S
0
2
4

6
8
10
12
14
bL
A
X1
S
bLAX2
S
bL
AX3
S
bL
A
X
4
S
bL
A
X
5
0
5
10
15
20
25
30

35
Sb
P
IN1
SbPIN2
S
b
PIN3
S
b
PIN4
Sb
PIN5
Sb
PIN
6
Sb
PIN
7
Sb
PI
N8
Sb
P
IN9
S
b
PIN1
0
SbPIN1

1
Sb
0
1
2
3
4
5
6
L
AX1
Sb
L
AX2
S
b
L
A
X
3
S
b
L
A
X
4
S
b
L
A

X
5
0
2
4
6
8
10
12
14
16
18
S
b
PGP1
S
bPG
P2
SbPGP3
S
bPG
P4
SbPGP5
S
bPG
P6
SbPGP7
S
bPG
P8

SbPGP9
Sb
PGP
10
SbPGP11
Sb
PGP
12
SbPG
P
13
Sb
PGP
14
SbPG
P
15
Sb
PGP
16
SbPG
P
17
Sb
PGP
18
SbPG
P
19
S

bPGP
2
0
SbPG
P
21
S
bPGP
2
2
SbPG
P
23
S
bPGP
2
4
0
10
20
30
40
50
60
SbPGP1
SbP
G
P2
S
bPG

P
3
SbPGP4
Sb
PG
P5
S
bPGP6
SbP
G
P7
Sb
PG
P
8
SbPGP9
S
bPG
P
10
S
bP
GP
1
1
SbPGP12
S
bPG
P
13

S
b
PGP
1
4
SbPG
P
15
S
bP
GP
16
S
b
PGP17
S
bPG
P
18
S
bP
GP
1
9
SbPGP20
S
bPG
P
21
S

b
PGP
2
2
SbPG
P
23
S
bP
GP
24
Leaf
Root
ABA treatment
ABA treatment
ABA treatment
Salt treatment
Salt treatment Salt treatment
Drought treatment
Drought treatment
Drought treatment
A
B
C
D
E
G
H
I
F

Fig. 6. Expression profiles of auxin trans-
porter genes SbPIN, SbLAX and SbPGP
under ABA and abiotic stress conditions.
Seedlings at 3-weeks old were treated with
100 l
M ABA for 3 h, 150 mM NaCl for
7 days or no irrigation for 7 days. Real-time
PCR conditions were as in Fig. 3. (A), (B)
and (G) show expression levels of SbPIN,
SbLAX and SbPGP genes under ABA treat-
ment. (C), (D) and (H) show expression lev-
els of SbPIN, SbLAX and SbPGP genes
under salt treatment. (E), (F) and (I) show
expression levels of SbPIN, SbLAX and
SbPGP genes under drought treatment.
Auxin transporter gene families in Sorghum bicolor C. Shen et al.
2964 FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS
Materials and methods
Sequence retrieval and chromosomal mapping
The published genome annotations of S. bicolor were
downloaded from the DOE Joint Genome Institute (ftp://
ftp.jgi-psf.org/pub/JGI_data/phytozome/v4.0/Sbicolor/). The
SbPIN, SbLAX and SbPGP gene families were identified by
hidden Markov model (HMM) searches performed by the
program hmmer [69] on the downloaded proteome annota-
tions. Any protein containing a Mem_trans domain
(PF03547.11) was chosen, and after removing the PIN-like
proteins, the remaining proteins were considered SbPIN
proteins. AUX and LAX1–3 protein sequences in Arabidop-
sis were retrieved from the TAIR database (http://www.

arabidopsis.org) and used to build a HMM file for the LAX
protein family using hmmer. SbLAX proteins were identi-
fied by a HMM search against the proteome annotations of
sorghum, using the HMM file of the LAX protein family.
Any protein containing an ABC_tran domain (PF00005.20)
was extracted as a SbABC family member. SbPGP family
(SbABCB subfamily) members were assigned as suggested
previously [70]. Results were submitted to the Pfam data-
base () to confirm the candidate
sequences as SbPIN, SbLAX and SbPGP proteins, and to
determine protein domain architectures (E-value < 0.001).
Approximately 194 500 expressed sequence tag (EST)
sequences of S. bicolor were downloaded from the NCBI
EST database up to 27 August 2009. Only the top hits of
the blastn search results for each SbPIN, SbLAX and
SbPGP gene family member showing a bit score of 500 or
more were considered significant. Each family member in
this study was mapped to the sorghum chromosome accord-
ing to the position of genes in the GFF file in genome anno-
tation, and centromere information was based on the
sorghum genome [71]. Distinctive gene names were assigned
according to the position from the top to the bottom on
chromosomes 1–10. Information on duplicated segments in
the sorghum genome was determined by the SyMAP data-
base [72]. Visualization of chromosome and segmental
duplications was performed with the Circos tool [73].
Promoter analysis, multiple sequence alignment
and phylogenetic relationship analysis
Sequences of 2500 nucleotides before the start codon were
extracted from the genomic sequence of the SbPIN, SbLAX

and SbPGP gene families, and both strands were scanned for
cis-regulatory elements obtained from the literature using an
in-house perl script [50–52]. The muscle program [74] was
used for multiple sequence alignment. After manual editing
with jalview [75], the alignment file was used for phyloge-
netic analysis. Phylogenetic relationship analysis was
performed with mega 4 program [76], using the neighbor-
joining and maximum likelihood methods on the RAxML
webserver [77]. Maximum likelihood parameters were evalu-
ated using prottest 2.2 [78]. The transmembrane helices of
SbPIN, SbLAX and SbPGP proteins were predicted using
the TMHMM webserver ( />TMHMM).
Plant materials and growth conditions
S. bicolor L. Moench seeds were treated with 1% sodium
hypochlorite and after thorough washing, soaked for 2 days
for germination. Seedlings were grown in Murashige and
Skoog nutritional liquid medium for 3 weeks. Seedlings
were treated with 1 lm BR for 12 h, 10 lm IAA, 30 lm 1-
NOA, 25 lm NPA, 50 lm TIBA or 100 lm ABA for 3 h;
or 150 mm NaCl for 7 days. For drought treatment, germi-
nated sorghum seeds were planted in sand with Murashige
and Skoog liquid medium for 3 weeks, and not irrigated
for 1 week. Samples were taken from 3-week-old leaves,
stems, roots and young panicles, for tissue-specific expres-
sion analysis.
RNA isolation and real-time quantitative
RT-PCR analysis
Total RNA was extracted using the RNeasy Plant mini kit
(Qiagen, Hilden, Germany) according to the manufacturer’s
instructions. cDNA was synthesized using reverse transcrip-

tase M-MLV (Promega, Madison, USA). Quantitative real-
time PCR was performed in a LightCycler 480 (Roche) using
SYBR premix Ex Taq kit (TaKaRa, Dalian, China). Primer
pairs for individual gene families were designed with primer
express 2.0 software (Applied Biosystems, Foster city, CA,
USA). Sequences were confirmed using the blast program to
ensure amplification of unique and appropriate cDNA seg-
ments (Table S5). The specificity of reactions was verified by
melting curve analysis. The relative RNA levels for each of
gene were calculated from cycle threshold (C
T
) values accord-
ing to the DC
T
method (Applied Biosystems). SbACTIN
(Sb01g010030.1) was used as an internal standard. The
sequences of SbACTIN primer pairs were 5¢-ATGGC
TGACGCCGAGGATATCCA-3¢,5¢-GAGCCACACGGA
GCTCGTTGTAG-3¢. The qRT-PCR program was one cycle
of 95 °C for 10 s, folloowed by 45 cycles of 94 °C for 10 s,
60 °C for 10 s and 72 °C for 15 s. Quantification of each
cDNA sample was performed in triplicate.
Acknowledgements
We gratefully acknowledge Ping Lu, the Institute of
Crop Germplasm Resource in the Chinese Academy of
Agricultural Sciences for providing the Sorghum
bicolor (L.) Moench. The research was supported by
the National Natural Science Foundation of China
(Grant Nos 30770213 and 30971743), the National
C. Shen et al. Auxin transporter gene families in Sorghum bicolor

FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS 2965
High Technology Research and Development Program
of China (863 Program) (Grant Nos 2007AA10Z188,
2007AA10Z191 and 2008AA10Z125) and the Natural
Science Foundation of Zhejiang province, China
(Grant No. Y3080111).
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Supporting information
The following supplementary material is available:

Fig. S1. Multiple sequence alignment of the SbPIN
gene family.
Auxin transporter gene families in Sorghum bicolor C. Shen et al.
2968 FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS
Fig. S2. Multiple sequence alignment of the SbLAX
gene family.
Fig. S3. Multiple sequence alignment of the SbPGP
gene family.
Fig. S4. Exon–intron organization of the SbPIN,
SbLAX and SbPGP genes.
Table S1. Analysis of SbPIN, SbLAX and SbPGP gene
family members in Sorghum bicolor.
Table S2. Promoter analysis of SbPIN, SbLAX and
SbPGP genes in Sorghum bicolor.
Table S3. PIN, LAX and PGP genes in Arabidopsis.
Table S4. PIN, LAX and PGP genes in rice.
Table S5. Primer sequences of SbPIN, SbLAX and
SbPGP genes for real-time PCR.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
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should be addressed to the authors.
C. Shen et al. Auxin transporter gene families in Sorghum bicolor
FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS 2969