Planta (2007) 227:133–142

DOI 10.1007/s00425-007-0601-4

ORIGINAL ARTICLE

The expression of genes coding for auxin carriers in diVerent tissues and along the organ can explain variations in auxin transport and the growth pattern in etiolated lupin hypocotyls

M. Rocío Oliveros-Valenzuela · David Reyes · José Sánchez-Bravo · Manuel Acosta · Carlos Nicolás

Received: 7 May 2007 / Accepted: 23 July 2007 / Published online: 23 August 2007© Springer-Verlag 2007

Abstract Novel cDNA clones encoding putative auxininXux and eZux carriers have been isolated and character-ized from etiolated lupin (Lupinus albus L) hypocotyls. Thefull length of LaAUX1 and LaPIN1 and the partial length ofLaPIN3 were obtained and the deduced amino acid sequencerevealed a high degree of identity with the correspondingauxin carrier proteins from several species. The expressionof these genes depended on the tissue, the hypocotyl zoneand seedling age. LaAUX1 and LaPIN3 were expressed instele and outer tissues, while LaPIN1 was restricted to thestele. From the above-mentioned results and taking intoaccount the role proposed for the eZux carrier PIN1, it issuggested that LaPIN1 could mediate the basipetal auxintransport already described in this organ. LaAUX1 mightfacilitate auxin inXux in the transport cells. The expressionof the three genes decreased down the hypocotyl. The basip-etally decreasing gradient in the expression of LaPIN1 coin-cides with previous results showing a similar gradient in theintensity and polarity of auxin transport. The decisive roleascribed to PIN1 in polar auxin transport due to its localization

in the basal end of transporting cells and the existence ofsuch a gradient in the expression of LaPIN1 support thehypothesis of a barrier eVect (generated by decreasing auxintransport) previously proposed by our research group asbeing responsible for the auxin gradient, which controls thegrowth pattern in etiolated lupin hypocotyls.

Keywords Auxin eZux and inXux carriers · Auxin transport gradient · Etiolated hypocotyl growth · Lupinus albus

AbbreviationsIAA Indoleacetic acidNPA 1-N-naphthylphtalamic acidPAT Polar auxin transportLaAUX1, LaPIN1, LaPIN3 Lupinus albus genes

Introduction

Auxin is involved in plant developmental processes such asgrowth, vascular diVerentiation, tropic responses, and theformation of lateral and adventitious roots, among others.Endogenous auxin indole-3-acetic acid (IAA) is transportedfrom cell to cell, and in shoots moves basipetally towardsthe roots in a process referred to as polar auxin transport(PAT). During recent years, more evidence for the hypothe-sis that PAT is restricted to certain cells and is mediated byinXux (AUX1 and LAX1-4 proteins) and eZux carriers(PIN1-8 proteins) (Parry et al. 2001; Swarup et al. 2001;Friml et al. 2002; Benkova et al. 2003; Morris et al. 2004)has accumulated.

Recent immunolocalization studies have painted acomplex picture whereby the localization of both types of

José Sánchez-Bravo, Manuel Acosta and Carlos Nicolás contributed equally to the paper.

Nucleotide sequence database accession numbers: LaAUX1: AM235387; LaPIN1: AM235388; LaPIN3: AM407405.

M. R. Oliveros-Valenzuela · J. Sánchez-Bravo · M. AcostaDepartamento de Biología Vegetal (Fisiología Vegetal), Facultad de Biología, Universidad de Murcia, Campus de Espinardo, 30100 Murcia, Spain

D. Reyes · C. Nicolás (&)Departamento de Fisiología Vegetal, Centro Hispano-Luso de Investigaciones Agrarias, Facultad de Farmacia, Universidad de Salamanca, 37007 Salamanca, Spaine-mail: cnicolas@usal.es

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carrier is related with the direction of IAA transport and itsfunction in diVerent developmental events. Thus, AUX1might be involved in the formation of lateral roots since itwas detected in root primordia in diVerent developmentalstates (Marchant et al. 2002). The localization in diVerentcells (columella, cap and stele) of the root apex suggeststhat AUX1 is necessary in acropetal transport and rootgravitropism. As regards the PIN family, PIN1 is the bestcharacterized member and its pattern of distribution inshoot and root suggests a decisive role in PAT. Thus, PIN1was localized in the basal end of the xylem parenchymacells in shoot (Gälweiler et al. 1998; Noh et al. 2003) andprotophloem cells in root (Swarup 2001), which suggests arole in basipetal and acropetal transport, respectively. Uni-directional light produced a decrease in the basal localiza-tion of PIN1 on the dark side of Arabidopsis hypocotyls,which generated a radial gradient in the eZux carrier func-tionality and the subsequent auxin gradient and phototropicresponse (Blakeslee et al. 2004). PIN3 has been related tothe lateral auxin transport responsible for the tropicresponse due to its localization in the inner lateral side ofcortical cells in shoot and pericicle cells in root (Friml et al.2002). In contrast, PIN3 was seen to be uniformly distrib-uted in columella cells in vertical root and rapidly relocal-ized to the lateral side when the root was gravistimulated(Friml et al. 2002).

From the above-mentioned details, it can be deduced that,depending on the distribution of auxin carriers in cells andtissues, auxin can be directed to plant sites where auxin-inducible processes, such as the formation of leave primor-dia in the shoot apex (Stieger et al. 2002) or lateral root pri-mordia (Benková et al. 2003), occur. The distribution ofauxin carriers may also generate the auxin gradient responsi-ble for the subsequent growth gradient, as has been proposedin the phototropic response (Blakeslee et al. 2004). How-ever, there is almost no study on the role of these carriers innormal plant growth. Moreover, the participation of PAT innormal shoot growth is subject to controversy. Thus, it isgenerally accepted that PAT is involved in the growth ofseedlings growing in the light but not in those growing indarkness (Garbers et al. 1996; Lehman et al. 1996; Haga andIino 1998), although some studies have shown that the appli-cation of the PAT inhibitor, 1-N-naphthylphtalamic acid(NPA), inhibits growth in etiolated corn seedlings (Fellneret al. 2003). Unlike plants grown in the light, in which all thecells along the hypocotyl elongate continuously throughoutthe growth period (Shinkle et al. 1992; Gendreau et al.1997), there is a steep growth gradient in the dark with anelongation zone just below the apex (Ortuño et al. 1988;Gendreau et al. 1997). Consequently, if auxin from the apexis required for hypocotyl growth, PAT must provide it to thewhole hypocotyl, including the basal region, in light, and tothe apical elongation region in the dark.

In previous studies we have investigated the role of IAAand PAT in the growth of etiolated lupin hypocotyls. Theresults show that auxin from the apex appears to be respon-sible for hypocotyl growth, as decapitation of seedlingsstrongly reduced growth, which was restored after theapplication of exogenous IAA to the cut surface (Sánchez-Bravo et al. 1991). Etiolated lupin hypocotyls show a longi-tudinal growth gradient, which is matched by the endoge-nous auxin level (Sánchez-Bravo et al. 1986; Guerreroet al. 2001). Basipetal transport of IAA in etiolated lupinhypocotyls is inhibited by speciWc PAT inhibitors such as2,3,5-triiodobenzoic acid (Botía et al. 1992) and NPA(López-Nicolás et al. 2004), and during basipetal auxintransport, there is a lateral movement of IAA from the PATpathway in the stele to the outer, auxin-sensitive andgrowth limiting tissues (Sánchez-Bravo et al. 1991; LópezNicolás et al. 2004). Taken together, these results suggest arole for PAT in the growth of etiolated lupin hypocotyls,unlike that proposed for other species (Lehman et al. 1996;Jensen et al. 1998). Assuming that PAT provides the auxinfor growth, and taking into account that elongating growthis restricted to the apical region in etiolated hypocotyls(Ortuño et al. 1988; Gendreau et al. 1997), the questionremains as to how auxin accumulates in the elongationregion. In a former study, we proposed that variations inauxin transport along actively growing lupin hypocotylcould produce such accumulation (Sánchez-Bravo et al.1992). Recently we have extensively studied the variationof PAT along the lupin hypocotyls in seedlings of diVerentages and the results show that IAA transport was higher inapical (where most growth is localized) than in middle andbasal regions, although this longitudinal gradient tended todisappear with aging as hypocotyl growth slowed andWnally ceased (López-Nicolás et al. 2007). These resultssuggest that a basipetally decreasing gradient in PAT alongthe hypocotyl, which changes with age, may be responsiblefor the auxin distribution pattern controlling growth. Toinvestigate whether these PAT variations can be explainedin terms of auxin carrier distribution, in the present studywe Wrst conWrmed the presence of AUX1 and PIN genes inetiolated lupin hypocotyls and studied the expression ofthese genes in diVerent tissues along the hypocotyl in seed-lings of diVerent ages.

Materials and methods

Plant material

Seeds of lupin (Lupinus albus L.) cv Multolupa (kindlyprovided by Dr. J. M. Pozuelo from Centro de CienciasMedioambientales, CSIC, Madrid, Spain) were soaked for24 h in water and germinated in damp vermiculite at 25°C

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in darkness. After 3, 6 and 12 days samples of diVerent tis-sues and regions of the hypocotyls were collected, frozen inliquid nitrogen and stored at ¡80°C until use. According tothe time course curve of growth (see López-Nicolás et al.2001), 3 and 12 days correspond to the beginning and theend, respectively, of the linear growth period of etiolatedlupin hypocotyls. The whole hypocotyl was used in 3-day-old seedlings while in 6- and 12-day-old seedlings sectionsfrom the apical, middle and basal regions of the hypocotylwere analyzed. In the whole hypocotyl at 3 days and in theapical zone at 6 and 12 days, the outer tissues(cortex + epidermis) were separated from the inner tissues(stele i.e., pith + vascular cylinder) with a glass capillary(Fig. 1) as described by Sánchez-Bravo et al. (1991).

RT-PCR with degenerate primers and cloning of PCR product

Total RNA was extracted from the apical region of 6-day-old seedling of etiolated lupin hypocotyls using the RNAwiz(Ambion, Austin, TX, USA) following the manufacturer´sprotocol. cDNA was synthesized from 5 �g of RNA using1st Strand Synthesis kit for RT-PCR (Roche Diagnostics,Manheim, Germany) and oligo-p(dT) as a primer, followingthe manufacturer’s instruction.

This cDNA was used as a template to isolate DNA frag-ments encoding one inXux and two eZux auxin carriers byPCR, using a pair of degenerate primers based on regionsfound to be highly conserved in other AUXs and PINs[sense: 5�-CT(GT) CC(GC) TAT TC(GC) TTC (GT)C(GT)CAG TT(GT) GG(GT) ATG-3� and antisense: 5�-GCAT(GT)A TTT CCA C(GT)G T(GC)A C(AC)G CGT GTCC-3�] or PINs [sense: 5�-CC(AC) AAC ACT TTG GT(CT)ATG GGA AT-3� and antisense: 5�-GTC AT(AG) AC(AG)

CT(AT) G(CT) (ACT) GGA GGC AT-3�] described inplants. The PCR reaction for LaAUX1 cDNA clone wasperformed as follows: 94°C for 10 min, 40 cycles at 94°Cfor 1 min, 50°C for 1 min, 72°C for 1.5 min further 10 minat 72°C. The PCR reaction for LaPIN1 and LaPIN3 cDNAclones was performed as follows: 94°C for 10 min, 40cycles at 94°C for 1 min, 50°C for 1 min, 72°C for 2 minand further 10 min at 72°C. The ampliWcation productswith the expected size (726 bp for the auxin inXux carrier,835 and 843 for the auxin eZux carriers) were isolatedfrom 1% (w/v) agarose gel, cloned into pGEM-T Easy vector(Promega Corporation, Madison, WI, USA) and sequenced.

Isolation of the full-length cDNA clone

From the initial partial cDNA fragments, a full-lengthLaAUX1, LaPIN1 and a partial LaPIN3 cDNA clone wereproduced by using 5�and 3� rapid ampliWcation of cDNAends (RACE) to extend sequences. The RACE reactionswere carried out by using the BD Smart RACE cDNAampliWcation kit (BD, Palo Alto, CA, USA), following themanufacturer’s instructions. The products of RACE reac-tions were also cloned into pGEM-T Easy vector (Pro-mega).

DNA sequencing

Plasmid DNA templates were isolated by the Wizard PlusMinipreps DNA PuriWcation System (Promega). Determi-nation of the nucleotide sequence of the cDNA clone wasperformed on an ABI 377 sequencer (Perkin Elmer AppliedBiosystems, Inc. Foster City, CA, USA) using the dRhod-amine Terminator Cycle Sequencing Ready Reaction. TheDNA and deduced protein sequences were compared toother sequences in the EMBL (GenBank and SwissProtdatabases, respectively), using the FASTA algorithm (Pear-son and Lipman 1988).

Nucleic acid analysis

Genomic DNA from lupin hypocotyls was extracted usingthe Plant DNA Isolation kit (Roche Diagnostics) followingthe manufacturer’s recommendations. For Southern-blotanalysis, DNA (5 �g) was digested using diVerent endonuc-leases, fractionated on a 0.8% agarose gel, and blotted ontoHybond N nylon membranes (Amersham, Buckingham-shire, UK) according to the manufacturer’s instructions andhybridized with speciWc probes obtained from the 3´non-coding region of the three diVerent cDNA clones in order toavoid a cross hybridization with other AUX or PIN from L.albus. The probes were labeled with 32P using the Redi-prime II Random Prime Labelling System kit (Amersham)by overnight incubation at 65°C in 5X SSPE [NaCl

Fig. 1 Scheme showing the diVerent regions (A apical; M middle;B basal) and tissues (C + E cortex + epidermis; S stele) analyzed in anetiolated lupin hypocotyl. A glass capillary (Gc) was used to separatethe stele from the cortex + epidermis in the apical region

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(0.9 M), NaH2PO4.2H20 (50 mM), EDTA (5 mM), pH 7.7,SDS (0.1%), Denhardt´s solution 5X [BSA (0.1% w/v),Ficoll (0.1%), PVP (0.1%)] and 250 mg/ml of deoxyribo-nucleic acid from salmon testis (ssDNA). The membraneswere washed at 65°C with 3X SSPE, 0.1 % SDS for15 min; 2X SSPE, 1X SSPE, 0.5X SSPE and 0.1X SSPE,0.1 % SDS for 15 min. For Northern-blot, 10 �g of totalRNA isolated from diVerent tissues and zones of etiolatedlupin hypocotyls were fractionated in denaturing formalde-hyde agarose gels, transferred to nylon membranes andhybridized with the speciWc 32P-labeled probes. Finallymembranes were exposed and quantiWed by Phosphorim-ager analysis (Fujitsu, Kawasaki, Japan) using the Kodak 1DImage Analysis Software (Kodak, Rochester, NY, USA).

Results

Isolation and characterization of several cDNA clones from lupin hypocotyls encoding auxin carriers

Total RNA from the apical zone of 6-day-old etiolated lupinhypocotyls were used for RT-PCR ampliWcation usingdegenerated oligonucleotides corresponding to the conservedsequences among AUX and PIN proteins described in otherplant species. Thus, three cDNA fragments were obtainedand, by means of RACE extension, the full-length cDNAclones of two of them were obtained (LaAUX1 and LaPIN1).The sequences of the three cDNA clones were registered inthe EMBL and Gene Bank Nucleotide Sequences Databasesunder the following accession numbers and compared:

AM235387: This full-length cDNA clone was 1635-bplong and contained an ORF of 1398 bp. Comparison of thededuced amino acid sequence with EMBL databasesrevealed high identity with diVerent proteins of the AUXfamily and hence, this clone was named LaAUX1. Thehighest identity was found with the AUX family of auxininXux carriers from Medicago truncatula (98.7%), Arabid-opsis thaliana (96.2%) and Populus tremula x Populustremuloides (94.6%) (data not shown).

AM235388: This full length cDNA clone was 2167-bplong and contained an ORF of 1821 bp. Comparison of thededuced amino acid sequence with EMBL databasesrevealed high identity with diVerent proteins of the PINfamily of auxin eZux carriers. The highest identity wasfound with PIN carriers from Momordica charantia(76.7%), Populus tremula x Populus tremuloides (78%)and Cucumis sativus (73.7%). Finally, due to the high iden-tity with AtPIN1 gene from Arabidopsis (69%) this clonewas named LaPIN1 (data not shown).

AM407405: This partial cDNA clone was 2260-bp longand contained an incomplete ORF of 1569 bp. Comparisonof the deduced amino acid sequence with EMBL databases

revealed high identity with diVerent proteins of the PINfamily of auxin eZux carriers. The highest identity wasfound with PIN carriers from Populus tremula x Populustremuloides (75.9%) Populus tomentosa (73.8%) andPisum sativum (69.6%). Finally, due to the high identitywith AtPIN3 gene from Arabidopsis (66.4%) this clone wasnamed LaPIN3 (data not shown).

In order to check that LaPIN1 and LaPIN3 encode diVer-ent genes of the PIN family of auxin carriers both proteinswere aligned (Fig. 2). The identity of both PIN carriers wasfound to be around 60% but this identity was only 40% inthe central region, while they showed their highest identityin the N- and C- terminus.

Southern-blot analysis

Hybridization of the corresponding full-length LaAUX1,LaPIN1 and LaPIN3 with genomic DNA revealed, in allcases, several bands suggesting the presence of a multigenefamily for these genes in L. albus (data not shown). Whenthe genomic Southern-blot was performed using a speciWcprobe from the 3´non-coding region of all these genes, asingle band of hybridization [Fig. 3a (lane 3), b and c] wasobserved, indicating the speciWcity of the probe.

Expression of LaAUX1, LaPIN1 and LaPIN3 in etiolated lupin hypocotyls. Relation with hypocotyl growth

Northern-blot analysis of total RNA hybridized with a spe-ciWc probe from the LaAUX1 cDNA clone (Fig. 4), showedthat the expression of this mRNA transcript appeared in allthe diVerent zones and seedling ages analyzed. The highestlevel of expression was observed in the apical region of 6-day-old etiolated lupin hypocotyls (both in the outer and innertissues), coincident with the exponential phase of growth, andconsistent with the highest growth rate in this region at thisage. Additionally, it is worth noting that the levels of expres-sion of LaAUX1 transcripts were scarcely higher in the stelethan in the outer tissues (Fig. 4b). Finally, this cDNA clonetranscript was found to accumulate most in the apical regionindependently of the seedling ages, although the diVerencesamong regions were not very signiWcant (Fig. 4b).

Expression of LaPIN1 showed that, irrespective of age,the corresponding transcripts were essentially detected inthe stele while the transcript levels were almost undetect-able in the outer tissues. In 6- and 12-day-old seedlings, theexpression decreased down the hypocotyl (Fig. 5a, b).

The levels of expression of LaPIN3 were quite similar tothose observed for LaAUX1. Transcript levels weredetected in all the zones and seedling ages studied, mainlyin the stele (Fig. 6a, b), and again the highest levels ofexpression appeared in the apical region of 6-d-old etio-lated lupin hypocotyls.

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Discussion

Most studies on auxin carrier proteins have been carried outin Arabidopsis thaliana and its mutants although theirorthologs in many other plant species have been isolated

and characterized. However, the expression of AUX andPINs genes in dark-grown seedlings has been studied in areduced number of plant species including Arabidopsishypocotyls (Noh et al. 2003; Blakeslee et al. 2004), pea epi-cotyls (Chawla and DeMason 2004; Hoshino et al. 2005)and the transition zone between the hypocotyl and root ofcucumber (Kamada et al. 2003). In the present study wehave isolated and characterized from etiolated lupinhypocotyls novel cDNA clones coding for putative auxininXux and eZux carriers: the full-length cDNA clones ofLaAUX1 and LaPIN1 and the partial length of LaPIN3. Thededuced amino acid sequence revealed close identity withthe corresponding auxin carrier proteins from diVerentplant species. The study provides valuable new informationon diVerences in the expression of these genes dependingon the tissues, the hypocotyl zone and the age of seedlings.

In the available literature, there is no information on thetissular expression of genes encoding auxin carriers in etio-lated seedlings other than in Arabidopsis. We have studiedthe expression of these three genes in diVerent tissues of theelongation region of etiolated lupin hypocotyls: the wholehypocotyl in 3-day-old and the apical zone in 6- and 12-day-

Fig. 2 Alignment of the de-duced amino acid sequences of LaPIN1 and LaPIN3 from Lupinus albus L

Fig. 3 a–c Southern-blot analysis of genes LaAUX1 (a), LaPIN1 (b)and LaPIN3 (c). DNA (5 �g) from lupin hypocotyl was digested withNcoI (1), EcoRI (2) and BamHI (3) in a, NcoI (1) and EcoRI (2) in band HindIII (1) and EcoRI (2) in (c). M, � DNA cut with EcoRI andHindIII used as molecular size markers

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Fig. 4 a–b Expression of LaAUX1. a Northern-blot analysis of totalRNA isolated from diVerent tissues of 3-, 6- and 12-day-old etiolatedlupin hypocotyls. Cortex + epidermis (1) and stele (2) from the wholehypocotyl or the apical region, middle region (3) and basal region (4).RNA (10 �g per lane) was hybridized with LaAUX1 speciWc probe.Top ethidium bromide-satined gel showing rRNAs. b QuantiWcation ofhybridization signals obtained by using a phosphoimage scanner. Datawere normalized to the rRNA value. QuantiWed data are presented as a

stacked bar chart. The height of each bar represents the level of expres-sion in a given region as a percentage on the summation of the valuesmeasured in all the tissues and regions analyzed at the three ages. Thecircles show the expression in the diVerent tissues, while the numberscorrespond to the expression of LaAUX1 in the stele as a percentage onthe total measured in the apical region. See Fig. 1 for abbreviations oftissues and regions of the hypocotyl

Fig. 5 a–b Expression of LaPIN1. a Northern-blot analy-sis of total RNA isolated from diVerent tissues of etiolated lu-pin hypocotyls at diVerent ages. RNA (10 �g per lane) was hybridized with LaPIN1 speciWc probe. Lanes 1–4 and top gel as in Fig. 4. b QuantiWcation of the gene expression. Data were quantiWed, normalized and rep-resented as in Fig. 4. See Fig. 1 for abbreviations of tissues and regions of the hypocotyl

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old seedlings. The expression of LaPIN1 was restricted tothe stele (Fig. 5), where basipetal auxin transport is local-ized (Sánchez-Bravo et al. 1991; López-Nicolás et al.2004), which is in accordance with the localization of PIN1in the parenchymatous xylem cells of shoot and its involve-ment in PAT (Gälweiler et al. 1998; Noh et al. 2003).Unlike LaPIN1, the expression of LaAUX1 and LaPIN3occurred both in the stele and in the outer tissues, the valuesin the stele being slightly higher (Figs. 4, 6). AtPIN3 islocalized in the lateral side of cortical cells and has beenrelated with the lateral auxin transport occurring in photot-ropism and gravitropism (Friml et al. 2002). Its homologueLaPIN3 might be related with the lateral auxin transport(from the stele to the cortex and epidermis) alreadyobserved in etiolated lupin hypocotyls (Sánchez-Bravo et al.1991; López-Nicolás et al. 2004). This lateral transport mayprovide auxin to cells in the epidermis and outer cortex,which are the limiting factors in auxin-induced shootgrowth (Penny et al. 1972; Pearce and Penny 1983; Kutsc-hera et al. 1987). LaAUX1 in the stele and cortex couldfacilitate the inXux of auxin into the cells involved in basip-etal and lateral auxin transport, respectively.

The pattern of distribution of LaPIN1 and LaPIN3 agreeswith the model of auxin transport in etiolated lupin hypo-cotyl proposed in a previous study (López-Nicolás et al.2004). The model, based on the distribution of radioactivityobtained after application of labeled IAA to decapitatedseedlings (Sánchez-Bravo et al. 1991; López-Nicolás et al.2004), proposes that both basipetal and lateral auxin trans-port decreased from the apex to the base of the hypocotyls,which coincides with the variation in the expression of

LaPIN1 and LaPIN3, as shown in Fig. 7. Although the par-ticipation of LaPIN3 was not discarded, the incapacity ofNPA to inhibit the lateral auxin transport (López-Nicoláset al. 2004) raises some doubts on the mechanism of the lat-eral transport observed in lupin hypocotyls. In fact, there isno evidence to date that demonstrates the involvement ofPIN3 in the lateral distribution of auxin in normal growth.Unlike lateral transport, NPA eYciently inhibited basipetalauxin transport in etiolated lupin hypocotyls (López-Nicoláset al. 2004, 2007), as was to be expected from this well-known PAT inhibitor. Taking into account the important roleassigned to PIN1 in basipetal PAT due to its localization inthe basal end of transporting cells (Gälweiler et al. 1998), itis important to note the basipetal decrease in the expressionof LaPIN1 restricted to the stele (Fig. 5). This Wnding sup-ports the existence of the basipetally decreasing gradient inthe capacity to transport auxin demonstrated in previousstudies using isolated sections with diVerent localizationsalong the hypocotyl (Sánchez-Bravo et al. 1992; López-Nicolás et al. 2007). As discussed in these studies, the exis-tence of such a PAT gradient might generate the so-calledbarrier eVect, which could produce an auxin gradient alongthe hypocotyl, the auxin content being higher in the apicalelongation zone, as conWrmed in previous studies (Sánchez-Bravo et al. 1986; Guerrero et al. 2001). As regards the causeresponsible for the PAT gradient and the barrier eVect, otherfactors besides the LaPIN1 gradient must be taken intoaccount. For example, a gradient in Xavonoid compounds,which act as endogenous PAT inhibitors, have beendescribed (Brown et al. 2001; Peer et al. 2001). Auxin carri-ers involved in basipetal auxin transport, such as some phos-

Fig. 6 a–b Expression of LaPIN3. a Northern-blot analy-sis of total RNA isolated from diVerent tissues of etiolated lu-pin hypocotyls at diVerent ages. RNA (10 �g per lane) were used and hybridized with LaPIN3 speciWc probe. Lanes 1–4 and top gel as in Fig. 4. b QuantiWca-tion of the gene expression. Data were quantiWed, normalized and represented as in Fig. 4. See Fig. 1 for abbreviations of tis-sues and regions of the hypo-cotyl

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phoglycoproteins (PGP), are also expressed diVerently alongthe Arabidopsis hypocotyl and speciWc PIN-PGP pairingsinXuence PAT by modulating the rates of cellular auxinmovement (Blakeslee et al. 2007). Therefore, the resultshereby obtained support the hypothesis that the barrier eVectgenerates the auxin gradient responsible for the growth pat-tern in etiolated lupin hypocotyls.

The variation in the expression of LaAUX1, LaPIN1 andLaPIN3 along the hypocotyls at diVerent ages (Figs. 4, 5, 6)suggests a degree of growth dependence since, as a rule, thevariation in the expression was parallel to the growth varia-tion described by Sánchez-Bravo et al. (1986). Thus, in 6-and 12-day-old lupin seedlings the expression decreasedfrom the apex to the base of hypocotyl, which agrees withthe results obtained in etiolated pea epicotyls, where theexpression of PsPIN1, PsAUX1 and PsPIN2 (belonging tothe same subfamily as AtPIN3) was higher in the elongationzone than in the basal zone (Hoshino et al. 2005). This lon-gitudinal variation in the expression of LaAUX1, LaPIN1and LaPIN3 coincides not only with the variation in growthbut also with the variation in endogenous IAA content(Sánchez-Bravo et al. 1986; Guerrero et al. 2001) as well asthe variation in the intensity and polarity of auxin transport(López-Nicolás et al. 2007). The existence of basipetallydecreasing gradients in the IAA content, IAA transport andthe expression of genes encoding putative inXux and eZuxauxin carriers, suggest a relationship between the auxin and

its carrier proteins. In fact, several studies have conWrmedthe inXuence of auxin on the expression of the genes AUXand PINs. Thus, the expression was reduced in segmentsfrom etiolated Pisum sativum epicotyls after incubation inwater to reduce the auxin content, while subsequent treat-ment with IAA increased the expression as the auxin con-centration increased (Hoshino et al. 2005). Similar resultswere obtained with PINs from Arabidopsis thaliana (Peeret al. 2004) and AUX1 and PIN1 from Cucumis sativus(Kamada et al. 2003). Auxin also modiWed the cellularlocalization of these carriers, especially the PIN proteins,which maintain a cyclic traYc between the cell membraneand the endosome (Geldner et al. 2001). The PIN1 move-ment to (but not from) the cell membrane was inhibited bybrefeldine A, this inhibition being counteracted by auxinsbut not by other plant hormones (Paciorek et al. 2005). Theabove-mentioned particulars suggest that the synthesis andlocalization of auxin carriers is sensitive to auxin and there-fore, the auxin per se might modulate its own transport andpolarity, as proposed in the canalization hypothesis ofSachs (1988). This feedback regulation can contribute tothe maintenance of the PAT system necessary to establishthe auxin gradient responsible for developmental processsuch as the formation of root and leaf primordia, tropicresponses and shoot growth in the dark.

A local, PIN-dependent auxin gradient has been pro-posed to explain tropic responses (Blakeslee et al. 2004)

Fig. 7 a–c Similarity between the expression of LaPIN1 and the mod-el of auxin transport proposed by López-Nicolás et al. (2004). a Pictureshowing a 6-day-old etiolated lupin seedling. b Expression of LaPIN1in 6-day-old etiolated lupin hypocotyls. The level of expression in thediVerent tissues and regions as a percentage of the total in the wholehypocotyl is presented (data obtained from the assay in Fig. 5). c Pro-

posed model for auxin transport after local application of IAA to thestele of actively growing etiolated lupin hypocotyls. The size of verti-cal down arrows denotes the intensity of basipetal auxin transport. Lat-eral transport is also shown (horizontal arrows). Acropetal transport(vertical up arrows) in the outer tissues next to the glass capillary fol-lowing lateral auxin transport was also detected

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and as a common module in the formation of plant organssuch as lateral root primordia, cotyledon and leaf (Benkovaet al. 2003). The pattern of LaPIN1 expression reportedhere suggests that, besides the other factors describedabove, a PIN1 gradient might collaborate in the generation(through a barrier eVect) of the auxin gradient responsiblefor the growth pattern in etiolated lupin hypocotyls. ToconWrm this hypothesis, it will be necessary to know thedistribution of LaPIN proteins along the organ and theircellular localization. Such data will provide more informa-tion about the role of these auxin carriers in the growth ofetiolated lupin hypocotyls and experiments to this eVect arecurrently under way.

Acknowledgment This work was supported by the Ministerio deEducación y Ciencia, Spain and FEDER, EU (grant AGL-2004-07902)and a fellowship to M.R.O.-V.

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