Expression profile of PIN, AUX ⁄ LAX and PGP auxin 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 Jiang1 and YanHua Qi1 State Key Laboratory of Plant Physiology and Biochemistry, Zhejiang University, Hangzhou, China Department of Bioinformatics, Zhejiang University, Hangzhou, China James D Watson Institute of Genome Sciences, Zhejiang University, Hangzhou, China 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: 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 April 2010, accepted 10 May 2010) Auxin is transported by the influx carriers auxin resistant ⁄ 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 participate in specific organ development The expression profiles of these gene families were analyzed after treatment with: (a) the phytohormones indole3-acetic acid and brassinosteroid; (b) the polar auxin transport inhibitors 1naphthoxyacetic 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 similar 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 doi:10.1111/j.1742-4658.2010.07706.x Introduction Auxin plays a critical role in the spatiotemporal coordination of plant growth and development, through polar auxin transport [1–5] Auxin transport proteins in Arabidopsis are grouped into three families: auxin resistant ⁄ like aux1 (AUX1 ⁄ LAX) influx carriers, pin-formed (PIN) efflux carriers and P-glycoprotein Abbreviations ABA, abscissic acid; ABC, ATP-binding cassette; AUX1 ⁄ LAX, auxin resistant ⁄ 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 C Shen et al (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, Gossypium 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 and AtPIN8 remains unknown [4,5,10–14] Reports on PIN genes in monocotyledons 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 transport 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 tillering [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 development [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 molecule indole-3-acetic acid (IAA) [19–21] The agravitropic 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-naphthylphthalamic acid (1-NPA) [22–25] AUX1 uses a novel trafficking pathway in plants that is distinct from PIN trafficking, and provides an additional mechanism for the fine regulation of auxin transport [26] The paralogs of AUX1, LAX1, LAX2 and LAX3 maintain phyllotactic patterning, and buffer the PIN-mediated patterning mechanism against environmental or developmental influences [27] The auxin influx carrier gene LAX3 is induced by auxin, and increased LAX3 activity reinforces the auxin-dependent induction of a selection 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 Auxin transporter gene families in Sorghum bicolor free endogenous auxin [29] The AUX1 ⁄ LAX family of auxin influx carriers is required for the establishment of embryonic root cell organization in Arabidopsis thaliana [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-dependent 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] Alternatively, 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 activation of phytochromes or cryptochromes [38] The PGP, PIN and AUX ⁄ LAX families independently transport auxin in both plants and heterologous systems However, PIN–PGP and AUX–PGP interactions also function both independently and coordinately 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 transport in specific tissues Similarly, an antagonistic effect is also observed in AUX1–PGP4 and AUX1–PGP1 coexpression [4,39–41] A newly developed Schizosaccharomyces 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 FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS 2955 Auxin transporter gene families in Sorghum bicolor C Shen et al 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 undergone 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 not [44] Of the 40 genes in this study, 26 were located in the duplication region by chromosome mapping The gene pairs in the duplication 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 duplicate 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 internalizational 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 asparagine [47] SbPIN1, -3, -4, -5 and -8 have a short hydrophilic 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 protein kinases such as PINOID kinase [45] Most SbPINs 2956 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 phosphorylated, 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 transmembrane 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 nucleotide-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 predicted 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 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 FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS C Shen et al Auxin transporter gene families in Sorghum bicolor Fig 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 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 predicted 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 (droughtinducible, ABRE-like element) [50], CACGTG (ABA-inducible) [51], and GNGGTG, GTGGNG and GAAAAA (salt-inducible) [52,53] The results of ciselement 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 analysis indicated that the ‘long’ PINs form groups 2–5, and group is comprised of the ‘short’ PINs: two AtPINs, four OsPINs and four SbPINs Group contains only one AtPIN protein (AtPIN1) compared with the three members (SbPIN6, -7 and -10) in sorghum, FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS 2957 Auxin transporter gene families in Sorghum bicolor A C Shen et al B C Fig Phylogenetic tree of the PIN, LAX and PGP families in Arabidopsis, rice and sorghum Gene families names in are black for Arabidopsis, 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] 2958 FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS C Shen et al 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 members, 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 and (Fig 2C, and Tables S3 and S4) SbPGP21 is close to AtPGP1, and two SbPGPs (SbPGP16 and SbPGP18) are close to AtPGP19 in group 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 phylogenetic trees (Fig 2A–C), and most genes, with one rice gene, formed individual sister pairs (1 : orthologous relationships) However, in group 3, a gene cluster was comprised of four SbPGPs (SbPGP10–13) and the OsPGP9 gene (n : orthologous relationship) In particular, 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 : orthologous 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 Auxin transporter gene families in Sorghum bicolor PCR was performed with total RNA from sorghum leaf, stem, root and flower Real-time RT–PCR analysis showed that expression of most SbPINs was constitutive 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 phenotype is complemented by strong expression of PaLAX1 (Prunus avium), causing multiple inflorescences [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 Similar 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, suggesting that SbPGP is like AtPGP, which functions in a tissue-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 synergistic interaction [59] Many auxin response genes are also regulated by BR [60–63] To determine if auxin transporters are also involved in phytohormone signaling, 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, FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS 2959 Auxin transporter gene families in Sorghum bicolor SbPIN1 20 SbPIN2 20 C Shen et al 10 5 100 0 L F S R SbPIN6 6000 5000 4000 3000 2000 1000 L F S R SbPIN11 50 40 30 20 10 L 100 F S R SbLAX5 35 30 25 20 15 10 300 200 L S R SbPIN7 F S R L F S R L F S R L F S SbPGP5 0.05 0.04 0.03 0.02 0.01 0.3 0.25 0.2 0.15 0.1 0.05 L F S R SbPGP10 L F S R SbPGP11 R SbPGP15 0.1 0.05 L F S R SbPGP20 0.4 0.3 0.2 0.1 L F S R S R SbPGP12 12 10 3.5 2.5 1.5 0.5 L F S R SbPGP16 L F S R SbPGP17 2.5 1.5 0.5 L F S R L SbPGP21 F S R SbPGP22 0.14 0.12 0.1 0.08 0.06 0.04 0.02 1.2 0.8 0.6 0.4 0.2 F S R SbPGP8 S R SbPGP9 0.04 0.02 L F S R SbPGP13 L F S R SbPGP14 L F S R SbPGP18 L F S R SbPGP23 14 12 10 L F S R SbPGP19 L F S R SbPGP24 0.3 0.025 0.25 0.02 0.2 0.015 0.15 0.01 0.1 R F 0.06 S L 0.1 F R 0.08 L S 0.2 L 0.03 F SbPGP4 0.4 F L 0.6 10 L 0.8 15 R SbLAX4 20 R SbPGP3 30 20 S 25 40 S SbPGP7 10 60 F R 20 80 L S 30 F 0.1 F S 0.2 L F 0.5 0.3 40 100 0.2 R SbPGP6 120 0.15 70 60 50 40 30 20 10 S R 1.5 0.4 0.01 L 2.5 0.5 0.03 F S 0.6 SbPGP2 0.04 L SbLAX3 L R 0.05 SbPGP1 R 0.05 R S 0.1 S F 0.15 F L 0.2 SbLAX2 10 L F SbPIN10 50 12 SbLAX1 L 200 0.5 0.5 SbPIN9 100 20 R R 0.02 S S 150 F F 10 1.5 L 0.1 15 40 0.2 20 SbPIN5 0.4 0.3 L SbPIN8 2.5 60 L 1.5 2.5 80 F SbPIN4 0.07 0.06 0.05 0.04 0.03 0.02 0.01 400 15 10 RNA relative level SbPIN3 500 15 0.005 0.05 0 L F S R L F S R L F S R Fig 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 weeks, from leaves, stems and roots Young panicles of sorghum were planted in Murashige 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 2960 FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS C Shen et al Auxin transporter gene families in Sorghum bicolor Many auxin influx ⁄ efflux carriers are induced by exogenous auxin, suggesting that changes in auxin concentration are mediated at both the tissue and cellular 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 phenotypically, for example, in the synergistic promotion of lateral root development by auxin and BR which increases acropetal auxin transport in Arabidopsis [59] 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 expression 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 upregulated by BR treatment in root (Fig 4D) SbLAX1 and SbLAX4 were downregulated by BR in leaf Under IAA treatment, several SbPGP genes, including 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 inhibited by BR treatment 35 30 25 20 15 10 A 10 IAA treatment B Leaf Root IAA treat ment 250 200 X5 A Sb L LA X4 X3 X2 A Sb Sb L X1 D Sb LA LA 20 BR treatment C Sb N Sb PI N Sb PI N 10 Sb PI N 11 Sb PI N Sb PI N N Sb PI Sb PI Sb PI N N PI PI Sb Sb Sb PI N N BR treatment 15 150 10 Sb LA X X X Sb LA X Sb LA Sb LA IAA treatment E Sb LA X Sb PI N Sb PI N Sb PI N Sb PI N Sb PI N Sb PI N Sb PI N Sb PI N Sb PI N Sb PI N 10 Sb PI N 11 20 P3 PG P Sb PG P5 Sb PG P Sb PG P Sb PG P8 Sb PG Sb P PG P Sb PG P Sb 1 PG P Sb PG P Sb PG P Sb 14 PG P Sb 15 PG P Sb 16 PG P Sb PG P Sb PG P Sb PG P Sb 20 PG P Sb 21 PG P Sb 22 PG P Sb PG P2 Sb Sb PG G P2 G Sb P 25 Sb P P1 F BR treatment 15 10 Sb PG P Sb PG Sb P2 PG Sb P3 PG P Sb PG Sb P5 PG Sb P6 PG P Sb PG Sb P8 PG Sb P9 PG Sb P10 PG Sb P11 PG Sb P12 PG Sb P13 PG Sb P14 PG Sb P15 PG Sb P16 PG P Sb 17 PG P1 Sb PG Sb P19 PG P Sb 20 PG Sb P21 PG Sb P22 PG P Sb 23 PG P2 Fig Expression profiles of auxin transporter SbPIN, SbLAX and SbPGP genes under IAA and BR treatment Total RNA was extracted from 3-week-old seedlings treated under indicated conditions The relative 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 lM IAA for h; BR treatment: lM BR for 12 h Real-time PCR conditions were as in Fig (A), (B) and (E) show expression levels 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 RNA relative level 10 FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS 2961 Auxin transporter gene families in Sorghum bicolor C Shen et al of the three gene families, SbPIN, SbLAX and SbPGP under NOA, NPA and TIBA treatment Surprisingly, the three families showed no distinct differences in transcriptional 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 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 100 1-NOA treatment A Leaf Root 1-NOA treatment 80 15 30 B 10 20 10 LA X LA X Sb LA X Sb LA X Sb X LA X LA Sb Sb LA X Sb LA X X5 L Sb X LA Sb A L A X Sb LA Sb LA X 11 N PI Sb 10 N Sb PI Sb PI N N PI Sb N PI N Sb Sb PI Sb PI N N Sb PI PI N Sb PI G Sb Sb PI N N X A 15 10 TIBA treatment F L 20 25 Sb 35 30 25 20 15 10 Sb TIBA treatment X 11 10 PI Sb Sb PI N N N Sb Sb PI N PI N N Sb Sb PI PI PI Sb Sb E 25 N N PI N PI Sb Sb PI N N PI Sb 30 4 10 20 NPA treatment 30 RNA relative level D 10 40 Sb Sb Sb LA X N PI N Sb PI 12 NPA treatment 50 Sb 11 10 N Sb PI Sb PI Sb PI N N N PI Sb Sb Sb PI C PI N N N PI Sb 60 Sb Sb PI PI N N 1-NOA treatment 20 15 10 H P3 PG P Sb PG P Sb PG P Sb PG P Sb PG P Sb PG Sb P9 PG P Sb 10 PG P1 Sb PG P Sb 12 PG P Sb 13 PG Sb P14 PG Sb P15 PG P Sb 16 PG Sb P17 PG P Sb 18 PG P Sb PG P Sb PG P Sb PG P Sb 2 PG P2 Sb PG P2 Sb NPA treatment Sb PG P Sb PG Sb P2 PG P Sb PG P Sb PG Sb P5 PG P Sb PG P Sb PG P Sb PG Sb P9 PG P Sb 10 PG Sb P11 PG P Sb 12 PG Sb P13 PG P Sb 14 PG P Sb 15 PG Sb P16 PG P Sb 17 PG P Sb 18 PG P Sb 19 PG Sb P20 PG P Sb 21 PG P Sb 22 PG Sb P23 PG P2 35 30 25 20 15 10 PG Sb PG Sb Sb PG P1 P2 I TIBA treatment Sb PG P Sb PG P Sb PG Sb P3 PG P Sb PG Sb P5 PG P Sb PG P7 Sb PG P Sb PG Sb P9 PG P Sb 10 PG P Sb 11 PG P Sb 12 PG P Sb 13 PG P Sb 14 PG Sb P15 PG P Sb 16 PG P Sb 17 PG P Sb 18 PG P Sb 19 PG P Sb 20 PG P Sb 21 PG Sb P22 PG P Sb 23 PG P2 40 35 30 25 20 15 10 2962 Fig Expression profiles of auxin transporter genes SbPIN, SbLAX and SbPGP under auxin transport inhibitor treatment Seedlings (3 weeks old) were treated with 30 lM 1-NOA, 25 lM NPA or 50 lM TIBA for h Real-time PCR conditions were as in Fig (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 FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS C Shen et al 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, especially in roots Many SbPGP genes were downregulated, 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 upregulated and 332 downregulated under TIBA treatment [14] However, we did not find an increased effect in these auxin transporter genes, even with a TIBA concentration twice that of NPA These results suggested that transcription of auxin transporter genes was controlled 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 implicated in abiotic stress responses [64–67] Various environmental 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 abiotic stress responses in sorghum, their expression profile 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 Auxin transporter gene families in Sorghum bicolor almost inhibited by all three treatments The expression level of SbLAX1, -2, -4 and -5 compared with SbLAX3 in leaves was lower than in roots when treated 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 cryptochromes [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), suggesting 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 morphogenic response in Arabidopsis roots [68] Moreover, in the auxin transporter mutant aux1–7, the lateral root proliferation component of the salt stress-induced morphogenic response is completely abrogated This provides genetic and physiological evidence that the auxin influx carrier is involved in the response to salt stress To understand the relationship between auxin transporter and abiotic stress in detail, a combination of molecular biology, reverse genetics and plant physiology may help to identify the biological function of each transporter For example, loss- or gain-of-function mutants of auxin transporters can be obtained through T-DNA insertion or activation–tagging methods 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 FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS 2963 Auxin transporter gene families in Sorghum bicolor 25 G 20 Sb L A A X X Sb L A X Sb L A Sb L L A A X5 X4 A Sb Sb L X LA Sb X X X5 X4 A A Sb L A X A X3 Drought treatment F Sb L A L Sb X X A Sb L Sb L Salt treatment D Sb L 11 N PI Sb 10 N Sb PI N Sb PI N Sb PI N Sb PI N Sb PI N Sb PI Sb P IN Sb P IN Sb PI N N E 14 12 10 Sb PI N Drought treatment 35 30 25 20 15 10 Sb PI RNA relative level Sb PI N Sb PI N Sb PI N Sb PI N Sb PI N Sb PI N Sb PI N Sb PI N Sb PI N 10 Sb PI N 11 Salt treatment X C A Leaf Root ABA treatment B Sb L Sb PI N Sb PI N Sb PI N Sb PI N Sb PI N Sb PI N Sb PI N Sb PI N Sb PI N Sb PI N 10 Sb PI N 11 A Sb L 3.5 2.5 1.5 0.5 ABA treatment 60 50 40 30 20 10 C Shen et al ABA treatment 15 10 Sb PG P Sb PG P Sb PG P Sb PG P Sb PG P5 Sb PG P Sb PG P Sb PG P8 Sb PG Sb P9 PG P Sb 10 PG P Sb 11 PG P1 Sb PG P Sb 13 PG P Sb 14 PG P Sb 15 PG P Sb 16 PG P1 Sb PG P Sb 18 PG P Sb 19 PG Sb P20 PG P Sb 21 PG P Sb 22 PG P Sb 23 PG P2 18 16 14 12 10 Salt treatment Sb PG P Sb PG Sb P2 PG P Sb PG Sb P4 PG P Sb PG Sb P6 PG P Sb PG Sb P8 PG Sb P9 PG P Sb 10 PG Sb P11 PG P Sb 12 PG Sb P13 PG P Sb 14 PG Sb P15 PG P Sb 16 PG P Sb 17 PG P Sb 18 PG Sb P19 PG P Sb PG P Sb 21 PG P2 Sb PG Sb P23 PG P2 H 60 I 50 40 Drought treatment 30 20 PG Sb Sb P1 PG P Sb PG P Sb PG P4 Sb PG P Sb PG P6 Sb PG P Sb PG P8 Sb PG Sb P9 PG P Sb 10 PG P Sb 1 PG P Sb 12 PG P Sb 13 PG P1 Sb PG P Sb 15 PG P Sb 16 PG P1 Sb PG P Sb 18 PG P Sb PG P2 Sb PG P Sb 21 PG P Sb 2 PG P Sb 23 PG P2 10 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 2964 Fig Expression profiles of auxin transporter genes SbPIN, SbLAX and SbPGP under ABA and abiotic stress conditions Seedlings at 3-weeks old were treated with 100 lM ABA for h, 150 mM NaCl for days or no irrigation for days Real-time PCR conditions were as in Fig (A), (B) and (G) show expression levels of SbPIN, SbLAX and SbPGP genes under ABA treatment (C), (D) and (H) show expression levels 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 the role of auxin transporters in mediating cross-talk between the auxin response and various abiotic stresses The action of auxin transporters under cross-talk regulation will be an important subject for detailed future studies FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS C Shen et al 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 annotations 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 Arabidopsis 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 identified 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 database (http://pfam.sanger.ac.uk) 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 according to the position of genes in the GFF file in genome annotation, 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 database [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 phylogenetic analysis Phylogenetic relationship analysis was performed with mega program [76], using the neighborjoining and maximum likelihood methods on the RAxML Auxin transporter gene families in Sorghum bicolor webserver [77] Maximum likelihood parameters were evaluated using prottest 2.2 [78] The transmembrane helices of SbPIN, SbLAX and SbPGP proteins were predicted using the TMHMM webserver (http://www.cbs.dtu.dk/services/ TMHMM) Plant materials and growth conditions S bicolor L Moench seeds were treated with 1% sodium hypochlorite and after thorough washing, soaked for days for germination Seedlings were grown in Murashige and Skoog nutritional liquid medium for weeks Seedlings were treated with lm BR for 12 h, 10 lm IAA, 30 lm 1NOA, 25 lm NPA, 50 lm TIBA or 100 lm ABA for h; or 150 mm NaCl for days For drought treatment, germinated sorghum seeds were planted in sand with Murashige and Skoog liquid medium for weeks, and not irrigated for week Samples were taken from 3-week-old leaves, stems, roots and young panicles, for tissue-specific expression 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 transcriptase M-MLV (Promega, Madison, USA) Quantitative realtime 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 segments (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 (CT) values according to the DCT 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 FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS 2965 Auxin transporter gene families in Sorghum bicolor C Shen et al 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) References Friml J (2003) Auxin transport – shaping the plant Curr Opin Plant Biol 6, 7–12 Kepinski S & Leyser O (2005) Plant development: auxin in loops Curr Biol 15, R208–R210 Woodward AW & Bartel B (2005) Auxin: regulation, action, and interaction Ann Bot 95, 707–735 Petrasek J, Mravec J, Bouchard R, Blakeslee JJ & Abas M (2006) PIN proteins perform a rate-limiting function in cellular auxin efflux Science 312, 914– 918 Tanaka H, Dhonukshe P, Brewer PB & Friml J (2006) Spatiotemporal 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Zardoya R & Posada D (2005) ProtTest: selection of best-fit models of protein evolution Bioinformatics 21, 2104–2105 Supporting information The following supplementary material is available: Fig S1 Multiple sequence alignment of the SbPIN gene family FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS C Shen et al 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 Auxin transporter gene families in Sorghum bicolor 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 by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 2954–2969 ª 2010 The Authors Journal compilation ª 2010 FEBS 2969 ... 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. .. 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... 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 Auxin