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

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 a

transporter gene families in Sorghum bicolor under

phytohormone and abiotic stress

ChenJia Shen1, YouHuang Bai2,3, SuiKang Wang1, SaiNa Zhang1, YunRong Wu1,

Ming Chen1,2,3, DeAn Jiang1and YanHua Qi1

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

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: qyhjp@zju.edu.cn

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: dajiang@zju.edu.cn

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, SbLAX2and 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 posimi-lar 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

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.

(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

OsPIN1gene 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

LAX3is 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 CO2 [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 PGPgene 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

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

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,

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.

A B

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].

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

OsPGP9gene (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,

SbLAXand 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 sugginflorescenc-esting 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 SbACTINexpression, 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,

0 1 2 3 4 5 6

SbPIN1

0

5

10

15

0 5 10 15

0 0.01 0.02 0.03 0.04 0.05 0.06

0 0.1 0.2 0.3 0.4

SbPIN6

0

1000

2000

3000

4000

5000

0 1 2 3 4 5 6

0 0.5 1 1.5 2

0 5 10 15

0 50 100 150 200

SbLAX1

0 5 10 15 20 25 30 35

SbLAX2

0 2 4 6 8 10

0 0.05 0.1 0.15

0 0.5 1 1.5 2 2.5 3

SbLAX5

0

20

40

60

80

0 0.5 1 1.5 2

0 0.01 0.02 0.03 0.04

0 0.1 0.2 0.3 0.4 0.5

0 0.2 0.4 0.6 0.8 1

SbPGP5

0

0.01

0.02

0.03

0.04

0 5 10 15 20 25

0 0.02 0.04 0.06 0.08 0.1

SbPGP10

0

0.05

0.1

0.15

0.2

0.25

0 20 40 60 80 100

0 1 2 3 4

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 4 6 8 10

0 0.5 1 1.5 2 2.5

0 0.2 0.4 0.6 0.8 1

0 2 4 6 8 10 12 14

SbPGP20

0

0.1

0.2

0.3

0 0.5 1 1.5 2 2.5 3

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

SbPGP6

0 10 20 30 50 60 70

SbPIN3

0 100 200 300 400 500

SbPGP7

0 10 20 30 40

L F S R

L F S R

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 Murashi-genes normalized to SbACTIN (Sb01g010030.1) Murashi-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.

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

SbLAX4were 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 SbP 0

Sb PIN1 1

0 2 4 6 8 10

SbLA

X 1

Sb LAX 2 SbLAX 3 SbLA

X 4 SbL AX 5

0 1 2 3 4 5 6

Sb P

GP 1

Sb P

GP 2 SbPGP 3 SbPGP4SbPGP5SbPGP6Sb P

G P7

Sb P

GP 8

Sb P

GP 9

Sb P

GP 10 SbPG P1 1 SbPG P1 2 SbPG P1 3 SbPGP14Sb P GP15

Sb P GP16

Sb P

GP 17 SbPG P1 8 SbPG P1 9 SbPGP20 SbPGP21Sb P GP22

Sb P

GP 23

Sb P

GP 24

Leaf Root

0 5 10

15 0

20 0

25 0

SbPIN 1 SbPIN 2 SbPIN 3 SbPIN 4 SbPIN 5 SbPIN 6 SbPIN 7 SbPIN 8 SbPIN 9

Sb PIN10 SbPIN11

0 5 10 15 20

Sb LAX1 Sb LAX2 Sb LAX3 Sb LA SbLAX5

0 5 10 15 20 25

SbPGP3SbPGP4SbPG P5

SbPGP7SbPG P8

SbPG

SbPG

SbPG P12 SbPG P13 SbPG

SbPG P15 SbPG P16 SbPG

SbPG P18 SbPG P19 SbPG

SbPG P21 SbPG P22 SbPG P23 SbPG P24

IAA treatment

t n m t a t A I t

n m t a t A I

BR treatment

BR treatment

BR treatment

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

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.

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

SbPGP2SbPGP3SbPGP4SbPGP5SbPGP6SbPGP7SbPGP8SbPGP9 SbPGP10 Sb PGP11 Sb PG

P12

Sb PGP13 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

0

10

20

30

40

50

60

SbPI

N1

SbPI

N2

SbP

IN3

SbP

IN4 SbP IN5 SbPIN6 SbPIN7 SbPI

N8 SbPI N9 SbPI N10 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 Sb PI

N2

SbP IN3 Sb PI

N4 SbP IN5 Sb PIN6 SbP IN

7 SbPIN8 Sb PI N9

Sb PIN10 SbPIN11

0 5 10 15 20 25 30 35

SbLAX 1 SbL AX2 SbL AX3 SbLAX4 Sb LAX 5

0

5

10

15

20

25

30

35

Sb PGP

1

Sb PG

P2

SbPGP3Sb PGP

4

Sb PG

P5

Sb PGP6 SbPGP 7

Sb PGP 8

Sb PG P9

Sb PG P1 0 SbP

G P11 SbPGP12 Sb PG P13 SbP

G P14 SbPGP15 SbPG P16 SbP

G P1 7 SbPGP18 SbPGP19 Sb PG P2 0 SbP

G P21 SbPGP22 Sb PG P23 SbP

G P2 4

0

10

20

30

40

SbPGP1SbPGP2Sb PG

P3

SbPGP4SbPG

P5 SbPGP6SbPGP7SbPGP8SbPGP9bPGP10 S S bPGP11 S bPGP12 bPGP13bPGP14 S S bPG

P1 5 bPGP16

S S bPGP17 S bPGP18 S bPGP19 bPGP20bPGP21 S S bPG

P2 2 bPGP23

S S bPGP24

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

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.

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 downreguupregu-lated 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, CO2, 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