Zhang-2012-The putative auxin efflux carrier O
The putative auxin ef?ux carrier OsPIN3t is involved in the drought stress response and drought tolerance
Qian Zhang 1,Jingjing Li 1,Wenjiao Zhang 1,?,Shuning Yan 1,?,Rui Wang 1,Junfeng Zhao 1,Yujing Li 1,Zhiguang Qi 1,Zongxiu Sun 2and Zhengge Zhu 1,2,*1
Hebei Key Laboratory of Molecular and Cellular Biology,College of Life Sciences,Hebei Normal University,Shijiazhuang,Hebei 050024,China,and 2
State Key Laboratory of Rice Biology,China National Rice Research Institute,Hangzhou,Zhejiang 310006,China
Received 30December 2011;revised 28July 2012;accepted 6August 2012;published online 15October 2012.*For correspondence (e-mail zhuzhengge@https://www.wendangku.net/doc/649671014.html,).?
These authors contributed equally to this work.
SUMMARY
The phytohormone auxin plays a critical role in plant growth and development,and its spatial distribution largely depends on the polar localization of the PIN-FORMED (PIN)auxin ef?ux carrier family members.In this study,we identify a putative auxin ef?ux carrier gene in rice,OsPIN3t ,which acts in auxin polar transport but is also involved in the drought stress response in rice.We show that OsPIN3t–GFP fusion proteins are localized in plasma membranes,and this subcellular localization changes under 1-N -naphthylphthalamic acid (NPA)treatment.The tissue-speci?c expression patterns of OsPIN3t were also investigated using a b -glucuronidase (GUS)reporter,which showed that OsPIN3t was mainly expressed in vascular tissue.The GUS activity in OsPIN3t pro::GUS plants increased by NAA treatment and decreased by NPA treatment.Moreover,knockdown of OsPIN3t caused crown root abnormalities in the seedling stage that could be phenocopied by treatment of wild-type plants with NPA,which indicated that OsPIN3t is involved in the control of polar auxin transport.Overexpression of OsPIN3t led to improved drought tolerance,and GUS activity signi?cantly increased when OsPIN3t pro::GUS plants were subjected to 20%polyethylene glycol stress.Taken together,these results suggest that OsPIN3t is involved in auxin transport and the drought stress response,which suggests that a polar auxin transport pathway is involved in the regulation of the response to water stress in plants.Keywords:OsPIN3t,auxin ef?ux carrier,drought stress,crown root,Oryza sativa .
INTRODUCTION
Plant growth and development are in?uenced by biotic and abiotic stresses and by endogenous phytohormones.In many cases,plants alter their endogenous hormonal levels to respond to environmental stresses.Auxin plays an essential role in regulating various aspects of plant growth and development,such as embryogenesis,cell division and elongation,vascular tissue differentiation,root patterning,shoot elongation,and embryonic pattern-ing (Leyser,2001,2006).Auxin also acts as a crucial signal in responding to abiotic stresses.There are two pathways involved in stress responses:auxin signaling and auxin transport.Several groups have reported that auxin sig-naling mediates stress responses in plants (Fukaki et al.,1996;Wyatt et al.,2002;Seo et al.,2009;Tognetti et al.,2010),while only a few reports address the auxin transport response under abiotic stresses (Shibasaki et al.,2009;Shen et al.,2010).
Drought stress is one of the major abiotic stresses that restrict plant growth and development.Many phytohor-mones,such as abscisic acid (Petrasek et al.,2006),salicylic acid (SA)and jasmonic acid (JA),are known to respond to drought stress (Xiong et al.,2002).In early reports,Davenport et al.(1977)demonstrated that the basipetal transport of auxin was reduced in cotyledonary petioles,resulting in earlier leaf loss under conditions of water de?ciency.The report revealed that auxin transport inhibitors and water de?cits had synergistic effects on leaf abscission.Moreover,osmotic stress caused a striking increase in basipetal polar auxin transport (Sheldrake,1979).Collectively,these results establish a link between responses to drought stress and polar auxin transport (PAT)in plants,but the molecular and cellular mechanisms involved in PAT regulating these responses under drought stress remain unclear.
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The Plant Journal a2012Blackwell Publishing Ltd
The Plant Journal (2012)72,805–816doi:
10.1111/j.1365-313X.2012.05121.x
Auxin is primarily synthesized in young plant tissues (Ljung et al.,2005),and PAT is mediated by a class of proteins called ef?ux carriers(Blakeslee et al.,2005;Zazi-malova et al.,2010).The regulation of polar transport observed for auxin in plant tissues appears to be unique to this hormone because this type of regulation has not been detected for other signaling molecules(Petrasek and Friml, 2009).Auxin is actively transported through in?ux and ef?ux carriers,which are positioned asymmetrically on the plasma membrane.A previous study suggested that at least two ef?ux carrier protein families exhibited auxin transport activity:the PIN and ABCB(B type ATP-binding cassette super family of transporters)families.The PIN proteins act as the major auxin ef?ux carriers in plants and show distinct polar subcellular localization,which determines the direc-tion of auxin?ux(Okada et al.,1991;Wisniewska et al., 2006).Similar to the PIN proteins,ABCB proteins are involved in cellular auxin ef?ux in both plant and heterolo-gous systems(Petrasek et al.,2006).The cooperation between the two types of auxin ef?ux proteins in auxin transport remains unclear(Mravec et al.,2008).
Eight PIN members have been identi?ed in the Arabidopsis genome,designated PIN1to PIN8.PIN1,PIN2,PIN4,and PIN7are plasma membrane(PM)-localized and strongly support PAT(Friml et al.,2002a,2003;Xu et al.,2006).PIN5, PIN6,and PIN8were shown to localize to the endoplasmic reticulum(ER)and were suggested to play a key role in the intracellular distribution of auxin and the regulation of cellular auxin homeostasis.
Although there are12putative auxin ef?ux carriers homologous to the AtPIN genes in the rice genome based on database searches,only OsPIN1(Xu et al.,2005)and OsPIN2(Chen et al.,2012)have been characterized in rice. For example,overexpression of OsPIN1was shown to partially reverse the inhibitory effect of NPA treatment at the seedling stage,and suppression of OsPIN1by RNA interference affected adventitious root development.Over-expression of OsPIN2resulted in the production of more tillers and reduced the inhibitory effect of NPA treatment. These?ndings indicate that OsPIN1and OsPIN2play distinct roles in the normal growth and development of rice and act as auxin ef?ux carriers.
We describe the isolation and characterization of a putative auxin ef?ux carrier in rice that shows high amino acid sequence identity to AtPIN3.The1857-bp full-length cDNA of this gene is designated OsPIN3t.The UniProtKB/ Swiss-Prot database indicates that OsPIN3has two tran-scriptional isoforms with lengths of1857bp[accession number D5A7J0(618amino acids)]and1770bp[accession number Q0JKX2(589amino acids)].In RT-PCR experiments, only the1857-bp full-length cDNA was found and isolated. We did not detect a1770-bp transcript of OsPIN3in different tissues of the japonica rice varieties Nipponbare and Zhonghua11under normal growth or drought-stress conditions.Overexpressing the1857-bp isoform of this gene under the35S promoter resulted in visible differences between plants under normal and drought stress conditions compared with controls,including more effective tillers, longer roots,a shorter shoot height and better growth.To provide evidence that OsPIN3t encodes an auxin ef?ux carrier,we used molecular and cellular approaches to demonstrate that OsPIN3t is an auxin-inducible gene.To observe the effect of NAA and NPA on OsPIN3t expression, we quanti?ed GUS activity in OsPIN3t pro::GUS rice lines following NAA or NPA treatment.We also addressed the possible link between auxin transport and drought stress. We measured the OsPIN3t transcriptional level in over-expression(OE)(OsPIN3t overexpressing)and RNA inter-ference(RNAi)(OsPIN3t knockdown)rice lines under normal and stress conditions and the GUS activity in OsPIN3t pro::-GUS rice plants under polyethylene glycol(PEG)stress or NPA treatment.Based on the obtained results,we postulate that OsPIN3t is directly induced in response to auxin and it is also involved in drought stress.
RESULTS
OsPIN3t encodes a PIN3family member
Bioinformatics studies suggest that OsPIN3t was presumed to be an auxin ef?ux carrier and a member of the PIN family. The OsPIN3t(AK063976)cDNA and genomic sequences comprise1857and4307nucleotides,respectively.The Uni-ProtKB/Swiss-Prot database suggested that OsPIN3t has two isoforms that are produced by alternative splicing(Figure S1 in Supporting Information).We performed repeated PCR ampli?cations of OsPIN3t using different Taq DNA polyme-rases but did not detect the1770-bp transcript.In this study, we only characterized the1857-bp transcript,which was ampli?ed using the primers P1and P2(Table S1).The OsPIN3t protein also showed conserved regions in its N-and C-termini with non-conserved regions between them. Hydropathy analyses of the OsPIN3t amino acid sequence showed that the protein contains three characteristic regions including two mem_trans superfamilies,?ve hydrophobic stretches in the N-terminus,a predominantly hydrophilic core,and a hydrophobic region with?ve transmembrane segments.These predicted results show similarity to OsPIN1b(Xu et al.,2005),and these domains are found in all PIN and PIN-like proteins.The OsPIN3t protein shares63% sequence identity with AtPIN3(At1g70940)and61% sequence identity with AtPIN4(At2g01420).In previous reports,OsPIN3t was referred to as OsPIN10a(Wang et al., 2009)or OsPIN3a(Miyashita et al.,2010).
OsPIN3t is responsive to auxin and auxin transport inhibitors
The promoter region of the OsPIN3t gene contains auxin-responsive elements(SURECOREATSULTR11and
806Qian Zhang et al.
a2012The Authors
The Plant Journala2012Blackwell Publishing Ltd,The Plant Journal,(2012),72,805–816
CATATGGMSAUR)(Xu et al.,1997;Maruyama-Nakashita et al.,2005).Speci?cally,there are two GAGACA sequences at positions)573and)745and two CATATG sequences at positions)167and)758in the promoter region(Figure S2). To test whether OsPIN3t is auxin inducible,several assess-ments were performed.First,we used quantitative RT-PCR to determine OsPIN3t transcriptional levels with NAA or NPA treatment at the seedling stage.Untreated seedlings showed an expected expression level,and the expression level was increased in NAA-treated plants and decreased in NPA-trea-ted plants(Figure1a;Table S1).We further analyzed the effect of NPA or NAA on OsPIN3t pro::GUS rice lines,in which the1846-bp upstream sequence(from ATG)was used to drive the GUS reporter gene.As expected,GUS activity increased strikingly with NAA treatment and decreased signi?cantly with NPA treatment(Figure1b).The GUS staining in the coleoptile of OsPIN3t pro::GUS rice lines strongly increased after treatment with50n M NAA and decreased distinctly after treatment with10l M NPA(Figure1c).Similar results were observed in Arabidopsis by another group(Tsuda et al., 2011).These results provide evidences that OsPIN3t is directly induced in response to auxin.
To assess the functional role of OsPIN3t in auxin trans-port,a genetic approach was used.Homozygous OE,RNAi and WT seeds were germinated in MS medium or MS medium supplemented with5l M NPA.After5days,more adventitious crown roots emerged from the WT and OE stems in MS medium,and fewer crown roots emerged from the plants subjected to NPA treatment(Figure2a,b).We counted the numbers of seminal and crown roots of1-to 9-day-old seedlings(Figure2c–f).The results suggested that knockdown of OsPIN3t suppressed the development of seminal and crown roots.To compare the root phenotypes and development of adventitious roots of OE,RNAi and WT plants,we used the T2generation in the following experi-ment.Two-day-old seedlings were transferred from MS medium to MS medium supplemented with5l M NPA or 0.2l M NAA.On the seventh day after transfer,the numbers of adventitious roots were counted.As shown in Table1, overexpression of OsPIN3t improved adventitious root growth,whereas knockdown of OsPIN3t suppressed adven-titious root growth.The decrease in adventitious roots observed in RNAi plants was phenocopied by NPA treatment of WT plants.This?nding shows that OsPIN3t is involved in root growth and development in rice(Inukai et al.,2005;Liu et al.,2005).
OsPIN3t is localized to the plasma membrane,and altered localization is observed under NPA treatment
As determined by the TMpred(Hofmann and Stoffel,1993) program,OsPIN3t contains10transmembrane domains.In Arabidopsis,PIN1,PIN2,PIN3,PIN4,PIN6,and PIN7share a similar molecular structure;these proteins exhibit a long central loop and are primarily localized to the plasma membrane.PIN5and PIN8possess a short central loop and localize to the internal cellular and plasma membranes. Therefore,the different PIN proteins show different subcel-lular localizations,which might be attributable to PIN-spe-ci?c molecular properties(Ganguly et al.,2010).OsPIN3t is expected to localize to the plasma membrane because it has a molecular structure similar to AtPIN3.To investigate the subcellular localization of OsPIN3t,three constructs, CaMV35S::OsPIN3t-GFP,OsPIN3t pro::OsPIN3t-GFP,and an empty CaMV35S::GFP vector,were individually introduced into EHA105cells,which were then transformed into tobacco and rice plants.We used confocal microscopy to examine the expression and subcellular localization of OsPIN3t–GFP fusion protein in the epidermal cells of tobacco and root tip cells of rice.Fluorescence microscopy showed that the OsPIN3t–GFP fusion protein was distributed
(a)
(b)(c)
Figure1.OsPIN3t responded to NAA or
1-N-naphthylphthalamic acid(NPA)treatment.
(a)Relative expression level of OsPIN3t in5-day-
old seedlings under10l M NPA or50n M NAA
treated for3h.Bars indicate the SE of three
replicates.
(b,c)The GUS activity and GUS staining
increased with50n M NAA treatment and
decreased with10l M NPA treatment in OsPIN3t-
pro::GUS rice lines.
OsPIN3t is involved in drought stress response807
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The Plant Journala2012Blackwell Publishing Ltd,The Plant Journal,(2012),72,805–816
only within the plasma membrane (Figure 3a,b)of tobacco epidermal cells and transgenic rice root cells.This result contrasted with what was observed for the GFP control,which showed ?uorescence throughout tobacco epidermal cells and rice root tip cells (Figure 3c).To further examine the expression and subcellular localization of OsPIN3t–GFP fusion protein in root tip cells of rice,plasmolysis of rice root epidermal cells expressing OsPIN3t–GFP fusion proteins was performed.Images also supported that the OsPIN3t localized to the plasma membrane (Figure 3d).
We further analyzed the effect of NPA treatment on the subcellular localization of OsPIN3t–GFP fusion protein in root tip cells of rice.Five-day-old rice root tips were treated with 10l M NPA;as shown in Figure 3(e),the plasma membrane localization of OsPIN3t-GFP fusion proteins was altered,and the proteins shifted to internal positions within cells.This ?nding suggests that NPA had an effect on the subcellular localization of OsPIN3t–GFP.
(a)
(b)
(c)(e)
(d)(f)
Figure 2.Plant phenotypes associated with OsPIN3t overexpression or OsPIN3t knockdown using RNA interference (RNAi).
(a)Five-day-old RNAi,wild type (WT),and overexpression (OE)rice seedlings grown in MS medium.All plants exhibited normal seminal roots;only the WT and OE plants presented crown roots.OsPIN3t knockdown suppressed crown root development.
(b)Five-day-old RNAi,WT,and OE rice seedlings germinated in MS medium supplemented with 1-N -naphthylphthalamic acid (NPA).The NPA treatment suppressed the development of crown roots in WT plants.
(c–f)Effects of OsPIN3t and NPA treatment on root development.The numbers of seminal and crown roots in WT,OE,and RNAi rice plants were counted from the ?rst to the ninth day.
Plots (c)and (e)represent untreated plants while (d)and (f)are standards for NPA treatment.Error bars indicate the SE of the three independent experiments.More than 15transgenic rice plants were used in each experiment.
Table 1Number of roots in wild type (WT),overexpression (OE)and RNA interference (RNAi)seedlings with NAA or 1-N -naphthylph-thalamic acid (NPA)treatment
Genotypes Number of adventitious roots
WT OE
RNAi Untreated 8.75?1.30b 10.25?0.43a,b
6.75?0.83c
NAA 9.25?0.83b 11?0.71a 9?0.71b NPA
5.5?0.50c
5.75?0.43c
5.5?1.11c
Means in the same column followed by the same letter are not signi?cantly different (P <0.05,least signi?cant difference test).a
Numbers present the mean ?SE of three separate experiments each performed on a population of 15 Seedlings grew for 9days under 0.2l M NAA treatment after a 2-day germination.c Seedlings grew for 9days under 0.5l M NPA treatment after a 2-day germination. Relative root number of WT,OE and RNAi seedlings with NAA or NPA treatment. 808Qian Zhang et al. a2012The Authors The Plant Journal a2012Blackwell Publishing Ltd,The Plant Journal ,(2012),72,805–816 Genetic analysis suggest that OsPIN3t confers tolerance to drought stress The1846-bp promoter sequence of OsPIN3t was analyzed using the PLACE database program(http://www.dna. affrc.go.jp/PLACE/)(Higo et al.,1999).The promoter sequence contains many putative stress response-related cis-acting elements,such as ABRE,CRT/DRE,and MYBRS (MYB recognition sites)elements(Figure S3). To examine the expression patterns of OsPIN3t and observe whether the OsPIN3t promoter is responsive to drought stress in rice,the OsPIN3t pro::GUS rice lines were used in the following experiment.Histochemical analysis of T1rice plants showed that the strongest signals were found in the coleoptile and vascular bundles of leaves, roots,and shoots,whereas weak signals were found in lamina joints.No GUS staining was observed in transgenic rice seeds or in?oral parts,except for the vascular bundles of the lemma and?lament(Figure4a–h).To assess whether OsPIN3t is involved in the drought stress response,GUS staining and GUS activity were monitored in OsPIN3t pro::GUS lines under PEG-induced osmotic stress(Lagerwerff et al.,1961).The GUS staining in coleoptiles and GUS activity in whole transgenic rice plants were greatly increased following exposure to20% PEG stress(Figure 4i,j). (a) (b) (c) (d)A B C D E F G H I J K L (e) Figure3.Subcellular localization of OsPIN3t–GFP in rice root tip of rice cells and tobacco epidermal cells. Treatment with1-N-naphthylphthalamic acid(NPA)affects OsPIN3t–GFP localization in the root tip of rice cells.The bright-?eld and merged images of rice cells show tissues stained with propidium iodide.Images in(a–d)were selected from at least three independent experiments,and three individual lines were used for each experiment.Bar=20l M.Transgenic rice root cells expressing OsPIN3t–GFP(A–F)and expressing GFP(G–I)before plasmolysis,and expressing OsPIN3t–GFP after plasmolysis(J–L). (a)Subcellular localization of OsPIN3t–GFP driven by the35S promoter in the5-day-old root tips of rice cells or transiently expressed in tobacco epidermal cells. (b)Subcellular localization of OsPIN3t–GFP driven by the OsPIN3t promoter in the5-day-old root tips of rice cells or transiently expressed in tobacco epidermal cells. (c)Subcellular localization of GFP alone driven by the35S promoter in the5-day-old root tips of rice cells or transiently expressed in tobacco epidermal cells. (d)PlasmolysisofricerootepidermalcellsexpressingOsPIN3t–GFPfusionproteins.Imagesareoverlayofpropidiumiodidestainingshowingcelloutlinesinredwiththe localization of the GFP fusions proteins in green.Images were taken of4-day-old root tips of rice following2h treatment with0.8M mannitol. (e)ToobservetheeffectofNPAontheOsPIN3t–GFPfusionprotein,rootsfromthecontrol(GFPalone)andOsPIN3t–GFPricelinesweretreatedwith10l M NPAfor30min. The images were obtained using the same confocal settings and are representative of15roots from three independent experiments. OsPIN3t is involved in drought stress response809 a2012The Authors The Plant Journala2012Blackwell Publishing Ltd,The Plant Journal,(2012),72,805–816 To further con?rm the change in the OsPIN3t transcrip-tional level in response to drought stress,we analyzed the OsPIN3t expression level in WT,OE and RNAi rice plants under drought stress conditions.We also tested the effect of 20%PEG treatment on seed germination in OsPIN3t OE and RNAi plants.Under normal conditions,there were no signi?cant differences in the seed germination rates or visible phenotypes of transgenic lines or WT plants; however,the OE plants exhibited longer roots(Figure5a; seedlings in1/2MS medium).We next placed the T2 generation of OE,RNAi and WT seeds onto1/2MS medium containing20%PEG(or medium without PEG as a control) and allowed the seeds to germinate and grow into seedlings. There was a noticeable difference between the seedlings grown under the PEG treatment(Figure5b).The OE seed-lings exhibited better shoot growth,longer roots,and a greater number of adventitious roots than the RNAi and WT plants.The growth of RNAi and WT plants was also inhibited by PEG treatment compared with the growth of OE plants. Using OsACTIN1as a reference gene,the OE lines showed higher OsPIN3t expression levels than the RNAi and WT plants(Figure5c).These data suggested that PEG-induced osmotic stress induced OsPIN3t expression.Moreover,the expression levels of two drought-responsive genes, OsDREB2A(Figure5d)and OsAP37,were higher in OE plants than in WT plants under stress conditions.Therefore, the overexpression of OsPIN3t led to up-regulation of OsDREB2A and OsAP37and increased tolerance of rice plants to drought stress(Kim and Kim,2009;Oh et al.,2009). To assay survival rates of rice plants under PEG-induced osmotic stress or water stress conditions,we did the followed experiments.First,10-day-old OE,RNAi and WT seedlings were transferred to1/2MS liquid medium con-taining20%PEG for7days and allowed to recover under normal conditions for another7days.The plants that did not grow after the recovery period were considered to be dead. At the end of the assay,more OE seedlings survived than RNAi and WT seedlings,which were greatly withered (Figure5e).Second,when OE,RNAi,and WT seeds germi-nated in soil,similar germination rates and almost the same rice plant seedling growth was shown for all three lines (Figure S4a).Last,the5-day-old seedlings were subjected to soil without the provision of water for6days,and the survival rates of the three lines were very different(Fig-ure S4b,c).Our data suggest that the OsPIN3t gene plays inter-related roles under drought stress conditions. (a)(b)(c)(d) (e)(f)(g)(h) (i)(j)Figure4.The OsPIN3t expression pattern in rice and the response to drought stress. (a–h)Histochemical analysis of OsPIN3t pro::GUS transgenic rice plants.Tissues from different stages were subjected to GUS staining:(a) germinated seed,(b)root and transverse section, (c)coleoptile,(d)stem and transverse section of a node,(e)lemma,(f)anther,(g)immature seed, and(h)mature seed.(i)Coleoptiles from3-day-old OsPIN3t pro::GUS seedlings were subjected to20%polyethylene glycol(PEG)stress for3h. (j)Three-day-old OsPIN3t pro::GUS seedlings were subjected to20%PEG stress for3h,and GUS activity was assessed.The error bars indi-cate the SE of three replicates.More than15 transgenic rice plants were used for each exper-iment(T9,T12,and T19represent three indepen-dent transgenic rice lines). 810Qian Zhang et al. a2012The Authors The Plant Journala2012Blackwell Publishing Ltd,The Plant Journal,(2012),72,805–816 Phenotypes of rice overexpressing OsPIN3t To better understand the function of OsPIN3t in rice,quan-titative RT-PCR was performed using WT,OE,and RNAi plants.The results showed that OsPIN3t expression was strongly increased in the shoot apex,shoot,root,and pani-cle in the independent OE lines but was signi?cantly decreased in the RNAi lines compared with the WT lines (Figure S5).Three independent homozygous RNAi lines (RNAi plants R3,R6,and R7)and three homozygous OE lines(OE plants OE3,OE8,and OE9)were used for further analysis.The results indicated that the OsPIN3t transcrip-tional levels were similar for the individuals of each of the three kinds of transgenic rice line(Figure S6).In the OE plants,several agronomic traits were different from in the WT plants,including signi?cantly increased root lengths and numbers of adventitious roots.In10days,the RNAi seed-lings exhibited shorter shoots and roots than the WT and OE plants(Figure6a–c).On the14th day after germination,the shoots and roots of RNAi seedlings were slightly longer and shorter,respectively,than those of WT plants,and the shoots of the OE plants were the shortest of the three types (Figure6d–f).These differences were maintained into the mature stage in the rice plants(Figure6g).The OE lines also presented more effective tillers and a higher average seed set percentage than the WT and RNAi plants(Figure6h,i). Although the OsPIN3t OE plants exhibited shorter shoots and slightly lower thousand kernel weights(TKWs),these plants presented more effective tillers and higher average rates of seed setting(Figure6h,i).The OE and WT plants showed similar seed and panicle phenotypes to WT and transgenic rice(Figure S7),and similar yields per plant under normal conditions;the RNAi plants showed fewer effective tillers,lower seed setting rates,lower TKWs, and a lower yield per plant compared with the WT and OE plants. To con?rm the visible phenotypic differences between OE and RNAi plants resulting from OsPIN3t gene overexpres-sion or knockdown,quantitative RT-PCR was performed using different primers(Table S1).We also repeatedly attempted semi-quantitative and quantitative RT-PCR with different cDNA templates,but we could not detect the1770-bp isoform of OsPIN3t. DISCUSSION The auxin response plays a crucial role in plant growth and development by forming local concentration gradients. Previous studies have revealed that the distribution of PAT ef?ux carriers is directional,allowing active?ow of auxin through plant tissues(Okada et al.,1991;Wisniewska et al., 2006).Auxin signaling has recently been reported to be mediated by environmental stress responses in plants (Ganguly et al.,2010;Jung and Park,2011;Rahman,2012; Tognetti et al.,2012).However,comparatively little insight has been obtained regarding the auxin transport response to drought stress,especially in rice.Our?ndings provide (a)(b) (e) (c) (d) Figure5.Overexpression of OsPIN3t leads to improved drought tolerance. (a)Six-day-old rice seedlings in1/2MS medium.With the exception of the longer roots of overexpression(OE)plants,the RNA interference(RNAi),wild type(WT), and OE plants exhibit almost identical phenotypes. (b)Rice seeds germinated in1/2MS medium supplemented with20%polyethylene glycol(PEG). (c,d)The relative expression levels of OsPIN3t and OsDREB2A under20%PEG treatment.Bars indicate the SE of three replicates. (e)Survival rates of OE,WT,and RNAi seedlings under20%PEG stress. OsPIN3t is involved in drought stress response811 a2012The Authors The Plant Journala2012Blackwell Publishing Ltd,The Plant Journal,(2012),72,805–816 evidence for the involvement of an auxin transport ef?ux carrier in the drought stress response. OsPIN3t encodes a member of the auxin ef?ux carrier protein family.By identifying the involvement of OsPIN3t in rice growth through PAT,we showed that:(i)overexpression of OsPIN3t improved drought stress tolerance in transgenic rice;(ii)when OsPIN3t pro::GUS plants were subjected to drought stress,GUS expression was responsive to drought stress,and GUS activity signi?cantly increased under NAA treatment and decreased under NPA treatment in the coleop-tiles of OsPIN3t pro::GUS rice plants;and(iii)OsPIN3t–GFP fusion proteins localized to the plasma membrane,and this subcellular localization was altered by NPA treatment. OsPIN3t expression is responsive to auxin or auxin transport inhibitor treatment There are four OsPIN genes onchromosome1:Os01g0643300, Os01g0919800,Os01g0715600,and Os01g0802700.The close (a) (b) (c)(d) (e)(f) (g) (h)(i)Figure6.Phenotypes of OsPIN3t transgenic rice plants. (a–d)Wild type(WT)and transgenic rice seed-lings on different days after germination.The overexpression(OE)shoot and root lengths were longer at10days after germination,but the OE shoot length was shorter compared with the other plants after10days’germination.Fourteen days after germination,the shoots of RNAi seedlings were slightly longer than those of WT and OE seedlings.Bar=2cm. (a)Two-day-old rice seedlings. (b)Five-day-old rice seedlings. (c)Seven-day-old rice seedlings. (d)Fourteen-day-old rice seedlings. (e)Shoot lengths of9-and14-day-old seedlings. (f)Root lengths of9-day-old seedlings. In(e and f)the shoot and root lengths from three independent experiments were measured(at least15seedlings for each).Error bars represent standard deviation.*P<0.05;**P<0.01. (g)The WT and transgenic rice plants at the ripening stage.(h)Seed setting rates of WT and transgenic rice.In(h),the error bars indicate the SE were determined using more than15plants for each line. (i)Effective tillers of WT and transgenic rice.In(i), the error bars indicate the SE were determined using more than15plants for each line. 812Qian Zhang et al. a2012The Authors The Plant Journala2012Blackwell Publishing Ltd,The Plant Journal,(2012),72,805–816 proximity of the chromosomal locations of the OsPIN genes suggests that the PIN gene family was generated by dupli-cation of chromosomal segments.The OsPIN proteins share conserved regions within their N-termini and C-termini,but their amino acid sequences diverge in central regions.These characteristics imply that different PINs mediate various functions in plant growth and development.In Arabidopsis, the divergent structures of PIN proteins are responsible for their different subcellular localizations and indicate their differential catalytic activities when involved in PAT (Ganguly et al.,2010).The subcellular localizations of rice PIN proteins have not yet been reported.In this study,we showed that OsPIN3t–GFP fusion proteins localize to the plasma membrane,and this localization is altered by NPA treatment(Figure3e),which suggests that the?ow and redistribution of auxin in rice root tips are mediated by OsPIN3t. Our results indicated that OsPIN3t was mainly expressed in vascular tissue(Figure4a–h),and OsPIN3t expression was up-regulated by NAA,without de novo synthesis of other proteins.The formation of vascular elements has been proposed to be regulated by PAT(Fukuda,2004).Previous studies indicated that OsPIN1b and OsPIN1d were expressed in presumptive vascular tissue areas and were associated with vascular bundle development(Wang et al.,2009). Moreover,overexpression of OsPIN3t in rice plant affected seedling growth rate in the roots and shoots.As per the Cholodny–Went hypothesis(1926),differential distribution of auxin in lateral directions due to gravity or light stimulation caused differential growth rates,which could ultimately lead to unbalanced growth or a loss of gravitropism in the shoot or root(Friml et al.,2002b;Ottenschlager et al.,2003;Brunoud et al.,2012).In addition,the?nding that GUS activity in OsPIN3t pro::GUS rice lines was promoted by NAA treatment and decreased by NPA treatment also strongly suggests that OsPIN3t is directly induced in response to auxin. OsPIN3t is involved in root growth at the seedling stage OsPIN3t participates in rice root development and plays a key role at the vegetative growth stage.Overexpression of OsPIN3t led to the development of longer roots and more adventitious roots in OE plants,whereas knockdown of OsPIN3t in rice plants resulted in slightly shorter adventi-tious roots.These data are consistent with the root pheno-types of Arabidopsis plants in which AtPIN3was knocked down(Friml et al.,2002b).Many studies have shown that lateral root formation in Arabidopsis and crown root for-mation in rice are regulated by auxin signaling and auxin transport(Marchant et al.,2002;Inukai et al.,2005;Liu et al., 2005).In this study,we monitored the seminal and crown root development of OE,RNAi,and WT plants under treat-ment with auxin or auxin transport inhibitors.The results indicated that the initiation of crown roots was altered by NPA and NAA treatments in OE and RNAi rice plants,which suggests that crown root development is controlled by auxin signaling through PIN proteins(Figure2c).In Arabidopsis,?ve PIN genes have been found to be associated with the control of cell division and cell expansion during root outgrowth(Blilou et al.,2005).Although the phenotypes associated with AtPIN and OsPIN3t overexpression cannot be compared because of the differences in root development between rice and Arabidopsis,these similarities suggest that OsPIN3t and AtPIN play similar roles in these two plant species. In this study,NPA treatment had little effect on root growth in the OE plants but had a greater impact in the RNAi plants.We deduced that higher levels of OsPIN3t expression could partially overcome the inhibition of adventitious root growth and development caused by NPA treatment (Table1).Taken together,these observations strongly imply that OsPIN3t is involved in root development.However, knockdown of OsPIN1resulted in signi?cant inhibition of the emergence and development of adventitious roots(Liu et al.,2005;Xu et al.,2005;Chen et al.,2012),and over-expression of OsPIN2did not result in induction of root differentiation in transgenic rice plants(Chen et al.,2012). These?ndings indicate that different OsPIN proteins might mediate diverse functions in root growth.It is therefore likely that extensive signaling regulates root development and PAT. OsPIN3t is involved in drought tolerance The function of AtPIN2in basipetal auxin transport was dramatically reduced by cold,and the function of AtPIN3in the root gravitropic response was also inhibited by cold stress.These data imply that cold stress affects auxin transport,rather than auxin signaling in Arabidopsis (Shibasaki et al.,2009;Shen et al.,2010).Drought stress affects the hormonal balance of a plant during growth and development by reducing cytokine synthesis and activating ABA biosynthesis(Haberer and Kieber,2002).Although the function of auxin in response to drought stress is relatively uncharacterized,water de?ciency was shown in early1977 to reduce the basipetal transport of auxin in cotton(Daven-port et al.,1977).In recent years,genomic and physiological data have revealed that plant hormones perform important actions within a complex network in which there is signi?-cant signaling(Nemhauser et al.,2006).The concentration of plant hormones is altered when rice plants experience water de?cits.Drought stress treatments signi?cantly reduced concentrations of indole-3-acetic acid in rice grains during the grain?lling stage(Yang et al.,2001).As the primary mediators of auxin transport in plants,PIN proteins were presumed to participate in the drought stress response either directly or indirectly.Phototropin1(phot1)(Wan et al.,2012)is an Arabidopsis ortholog of the Ser/Thr protein kinase PINOID(PID),which catalyzes PIN phosphorylation, contributes crucially to the regulation of apical-basal PIN OsPIN3t is involved in drought stress response813 a2012The Authors The Plant Journala2012Blackwell Publishing Ltd,The Plant Journal,(2012),72,805–816 polarity(Kleine-Vehn et al.,2009),and can improve drought tolerance at the seedling stage(Galen et al.,2007). There is currently no evidence for the direct involvement of other OsPIN proteins in the plant response to drought stress,probably because OsPIN family members share responsibilities related to plant growth,and most OsPIN genes are uncharacterized.Previous studies suggested that PEG osmotic stress could induce water de?cit stress in a relatively controlled manner(Lagerwerff et al.,1961;Yu et al.,2008;Huang et al.,2009),and our results showed OE OsPIN3t improving drought tolerance(Figures5and S4).In the drought stress experiments using transgenic rice plants, knockdown of OsPIN3t to lead to growth inhibition following 20%PEG treatment or water de?ciency.In contrast,over-expression of OsPIN3t resulted in better transgenic rice growth.To investigate whether OsPIN3t was involved in the drought stress response,we used OsPIN3t pro::GUS lines to examine GUS staining and activity when these lines were subjected to drought stress.As expected,GUS staining and activity signi?cantly increased under drought stress.To further con?rm that OsPIN3t affected the expression of other drought stress genes,we discovered that the rice drought-responsive genes OsDREB2A and OsAP37were signi?cantly up-regulated in OE plants and slightly up-regulated in RNAi plants(Figure5c,d).Importantly,studies have shown OsDREB2A expression to be induced by drought and salt stress(Dubouzet et al.,2003)and that the overexpression of OsAP37increased grain yield under drought stress condi-tions(Okada et al.,1991;Kim and Kim,2009;Oh et al.,2009). Moreover,overexpression of OsPIN3t in rice plants improved drought tolerance,which also suggests that OsPIN3t possesses distinct functions compared with OsPIN1 (Xu et al.,2005)and OsPIN2(Chen et al.,2012).Although we are uncertain about the mechanism of involvement of OsPIN3t in the drought stress response,OsPIN3t mediates auxin transport,plays an important role in rice root growth, and improves drought tolerance. In conclusion,OsPIN3t plays a key role in rice shoot and root development,is responsible for PAT,and is involved in drought stress responses. EXPERIMENTAL PROCEDURES Cloning of OsPIN3t and vector construction The rice(Oryza sativa L.)cultivar Zhonghua11was used for all experiments.The rice plants were grown in culture solution(Yosh-ida et al.,1976)in a growth room at temperatures of28/22°C(day/ night)with70%humidity under14/10h light and dark cycles.Total RNA was extracted from7-day-old seedlings,and a1857-bp open reading frame of OsPIN3t was ampli?ed with primers P1and P2via reverse transcription(RT)-PCR and ligated into a T-vector to form pMDPIN3t for sequencing and sub-cloning.For construction of the 35S::OsPIN3t-GFP vector,the full-length OsPIN3t cDNA without the stop codon was ampli?ed from pMDPIN3t.After veri?cation of the correct sequence,the fragment was cloned into the Kpn I and Xba I sites in the sense orientation,driven by a CaMV35S promoter in the pCAMBIA2300OCS expression vector.For the OsPIN3tpro::GUS vectors,the1846-bp DNA sequence upstream of the OsPIN3t ATG (start codon)was ampli?ed using primers P3and P4.After sequencing,the fragment was digested with Hin dIII and Xba I,and ligated into the pCAMBIA1300-GUS vector.The1857-bp coding se-quence with the stop codon was ampli?ed and digested with Bam HI and Xba I to construct the35S promoter::OsPIN3t::NOS overex-pression vector.To construct the RNAi vector,the424-bp coding sequence of OsPIN3t was ampli?ed with primers P5and P6,digested with Sac I and Spe I followed by Bam HI and Kpn I,and subsequently ligated into the pTCK303vector(Wang et al.,2004).All of the primer sequences used in these experiments are listed in Table S1.All constructs were introduced into the Agrobacterium tumefaciens EHA105strain to be used for rice transformation according to a published protocol(Hiei et al.,1994;Toki et al.,2006)with some modi?cations.Transformed calli were selected using hygromycin, and the regenerated transgenic rice plants were grown in a green-house under16/8h of light/dark,temperatures of28/25°C(day/ night),and80%relative humidity.The constructs containing GFP were also transformed into tobacco leaves via injection methods. Plant material and growth conditions The rice(O.sativa subsp.japonica)cultivar Zhonghua11was used as the plant material for the experiments described herein.Rice seedlings were grown in a growth chamber or greenhouse at tem-peratures of28/25°C(day/night)with a16/8h light/dark cycle under 50%relative humidity(Kawasaki et al.,2001).To assay primary agricultural traits,rice plants were also grown in?eld under normal conditions.To perform a drought tolerance assay,rice seeds were germinated in1/2MS medium containing20%PEG.For survival analysis,10-day-old rice seedlings were treated with20%PEG in1/2 MS liquid medium for1week,and the seedlings were then trans-ferred to new1/2MS medium for another week.The rice plants that did not grow were considered dead.For the NAA and NPA treat-ments,germinated rice seeds were sown in1/2MS solid medium containing0.5l M NAA or NPA and grown for5days. Quantitative RT-PCR Total RNA was extracted using TRI ZOL reagent according to the manufacturer’s instructions.For?rst-strand cDNA synthesis,500ng of total RNA and M-MLV reverse transcriptase(TaKaRa,DRR037A, https://www.wendangku.net/doc/649671014.html,/)was used.Quantitative RT-PCR was performed using the Applied Biosystems7000thermocycler(http:// https://www.wendangku.net/doc/649671014.html,/)with SYBR Premix Ex Taq following the reagent speci?cations and using primers speci?c for the examined genes(Table S1).OsACTIN1was used as an internal reference gene for normalization of all data in this experiment. Histological analysis of OsPIN3t promoter::GUS activity Histochemical GUS activity analysis was carried out according to Jefferson’s method(Jefferson et al.,1987).Transgenic rice plant samples were incubated in5-bromo-4-chloro-3-indolyl-b-glucu-ronic acid buffer in a dark37°C water bath.After staining,the tissues were rinsed several times with70%ethanol to remove chlorophyll and surface dyes.The GUS activity was quanti?ed by monitoring the cleavage of the GUS substrate4-methylumbelliferyl b-D-glucu-ronide(MUG).The results were averaged from three independent experiments. Subcellular localization of OsPIN3t To examine the subcellular localization of OsPIN3t,the CaMV35-S::OsPIN3t–GFP,OsPIN3t pro::OsPIN3t–GFP and CaMV35S::GFP 814Qian Zhang et al. a2012The Authors The Plant Journala2012Blackwell Publishing Ltd,The Plant Journal,(2012),72,805–816 vectors were constructed.All vectors were?rst introduced into the EHA105bacterial strain,followed by transient transformation into tobacco leaf epidermal cells;after3days of in?ltration,GFP expression was examined using a confocal laser-scanning micro-scope(Zeiss LSM510,https://www.wendangku.net/doc/649671014.html,/).All plasmids were also transformed into rice calli and transgenic rice plants to further analyze the subcellular localization of OsPIN3t fusion protein.Prior to imaging,roots were stained for30s in10mg ml)1propidium iodide(PI)in water.For the plasmolysis experiments,the roots were treated with0.8M mannitol for2h with shaking and next staining with10mg ml)1PI in0.8M mannitol. ACCESSION NUMBER The sequence data in this paper can be found in the GenBank/EMBL database(https://www.wendangku.net/doc/649671014.html,/),accession number of OsPIN3t is AK063976. ACKNOWLEDGEMENTS We thank Dr Kang Chong of the Institute of Botany,Chinese Academy of Science,for providing the pTCK303vector.Dr Xiang-dong Fu and Dr Yonghong Wang of the Institute of Genetics and Developmental Biology,Chinese Academy of Science,for provid-ing the pCAMBIA2300OCS vector and helping to analyze polar auxin transport.This work was supported by the National Natural Science Foundation of China(30870214),China National Major Scienti?c Project(2011ZX08009-003,2009ZX08009-017B),and the Natural Science Foundation of Hebei Province in China (C2010000385). SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1.Structures of two splicing isoforms of OsPIN3t based on the UniProtKB/Swiss-Prot database. Figure S2.Prediction of auxin-responsive elements in the promoter region of OsPIN3t. Figure S3.Prediction of cis-acting elements response to abiotic stress in the promoter region of OsPIN3t. Figure S4.Rice seedlings were subjected to water stress. Figure S5.Relative expression level of OsPIN3t in different rice tissues. Figure S6.Relative expression level of OsPIN3t relative expression level in different tissues of different transgenic rice lines. Figure S7.Phenotype of seeds and panicles of wild type and trans-genic rice. Table S1.Primers sequences used in plasmids construction and PCR. 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 ?les)should be addressed to the authors. 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