The B73 maize genome sequence has recently been completed and published (Schnable et al., 2009), and annotated CDS and filtered protein have also become available (release 5b.60). Two genome browsers are available at MaizeGDB¹ and MaizeSequence² websites. Putative maize auxin efflux carriers were initially identified by Blast searches against the reference genome using A. thaliana and O. sativa PIN transcript and protein sequences as queries (see Table S1 in Supplementary Material, for accession numbers of all the sequences used in this study). Following this approach, we retrieved the sequences of nine new maize PIN-related genes with their relative annotations (see Table S1 in Supplementary Material, for accessions). Additional Blast searches were performed to identify genomic survey sequences (GSSs), transcripts, and transcript assemblies (TAs) from PlantGDB³, from J. Craig Venter Institute⁴, from NCBI⁵, and from DFCI/TIGR database⁶. A large amount of genomic and expressed sequences was retrieved and univocally assigned to each corresponding primary identified sequence by alignment and consensus generation using ClustalX (Thompson et al., 2002), Geneious, and Lasergene DNAStar tools. In addition, GeneSeqer software⁷ was used to assign each TA to its corresponding genomic sequence and indirectly validate the splicing models of genome sequencing derived sequences. Redundant sequences were immediately discarded, while three PlantGDB GSSs without matching sequences in the genome database were further analyzed (AZM5_3988, AZM5_105354, and ZmGSStuc11-12-04.463.1). Two of these GSS (AZM5_105354 and AZM5_3988) resulted partial: the first corresponding to the 5′ end and the other to the 3′ end of a putative maize PIN gene and both showing high sequence similarity with a single PIN sequence of S. bicolor and, to a lesser extent, with Arabidopsis PIN2. Based on maize/sorghum alignment the two maize GSSs resulted as not overlapping and so the construction of a contig was not possible. However, using primer pairs designed on the putative 5′UTR and 3′UTR it was possible to amplify a single genomic and cDNA sequence from B73 and close the gap. This sequence and the third GSS (ZmGSStuc11-12-04.463.1) are not included in the B73 genome databases.

As described for the two partial GSS, several primers (Table S2 in Supplementary Material) were designed on the retrieved full-length sequences and were then used in PCR experiments using B73 inbreed genomic DNA and cDNA as template to directly amplify ZmPIN genes and further validate them. Cloning of the PCR amplification products was performed with the pCR^®II-TOPO^® TA-Cloning kit (Invitrogen) or with the pGEM^®-T Easy Vector Systems (Promega). E. coli competent cells were transformed by heat-shock and selected on plates containing LB medium, plus ampicillin, and X-Gal (IPTG was added when necessary). Plasmids were then purified using the Plasmid Mini Kit (QiAgen), following the manufacturer’s instructions. After sequencing on both strands, cloned sequences were edited, aligned against the reference genome, and translated to putative protein sequences using Geneious and Lasergene DNAStar softwares.

Despite many attempts, the ZmPIN10b full-length cDNA sequences resulted as being un-amplifiable from any of the tissues used in our study, while the genomic sequence was easily amplified and cloned. Curiously no ESTs or TAs corresponding to this gene were found in public databases, suggesting that this gene could be expressed at very low levels or in extremely specific and restricted domains/cell-types (see also the genome-wide atlas of gene expression data reported in Figure S5 in Supplementary Material; Sekhon et al., 2011). A similar situation concerns the ZmPIN5b locus. Annotation on genome browser shows a stop codon soon after the junction between the first and second exon, resulting in a truncated protein. Anyway the alignment between ZmPIN5b genomic sequence and sorghum ortholog CDS seems to indicate that the CDS predicted in the maize annotation could be wrong: a single base change in the first exon/intron results in the skipping of stop codon and in the “in silico” translation of a functional protein homolog to PIN5 of different species. Unfortunately just the first exon was successfully amplified from cDNA, while several primers designed from the second to the fifth exon sequences did not work in PCR amplifications. As for ZmPIN10b, no EST corresponding to ZmPIN5b were retried from databases and no expression has been detected by a genome-wide transcriptome analysis (Figure S5 in Supplementary Material; Sekhon et al., 2011), making it impossible to verify the actual CDS sequence of this gene. Studies described below (phylogenetic and protein profiles analyses) were done using the full-length protein sequence.

The phylogenetic relationships between newly identified maize PIN proteins and PINs of eudicots and monocots were determined through a robust phylogenetic analysis which also allowed determination of the evolution of the PIN auxin efflux carriers family from early-land plants (mosses) to monocots and eudicots. PIN protein sequences of A. thaliana and Medicago truncatula (two herbaceous eudicots), Populus trichocarpa and Vitis vinifera (two arboreal eudicots), Z. mays, O. sativa, S. bicolor, Brachypodium distachyon, and Setaria italica (five monocots), and Physcomitrella patens and Selaginella moellendorffii (a bryophyte and lycophyte, respectively) were retrieved by family blast searches in the Phytozome v7.0 database⁸. All the accessions of the sequences employed in this study are reported in Table S1 in Supplementary Material. Given the large amount of sequences, a subset of “pocket” trees was generated excluding PIN proteins of M. truncatula, V. vinifera, S. bicolor, and S. italica (Figure 1; Figures S2 and S3 in Supplementary Material), while a poster tree with all the sequences is reported in Figure S1 in Supplementary Material.

Evolutionary models of PIN proteins were performed using three different approaches and the results compared. First PIN proteins were aligned with ClustalX 1.81 (Blosum Weight Matrix; Gap Opening Penalty: 5; Gap Extension Penalty: 0.20; Thompson et al., 2002), and a neighbor-joining phylogenetic tree was prepared with Phylip package (Felsenstein, 1989) using 100 bootstraps. In this case the yeast PIN-like protein AEL1 (NP_593365 from Schizosaccharomyces pombe) was included in the analysis as outgroup (Yang and Murphy, 2009; Zazimalova et al., 2010). The phylograms were drawn using Tree-View 1.6.6⁹ and reported in Figure 1 and Figure S1 in Supplementary Material. In addition, PIN protein sequences were aligned with MAFFT algorithm (Katoh et al., 2002, 2009) available at the EMBL-EBI website¹⁰. Phylogeny reported in Figure S3 in Supplementary Material was inferred with the Molecular Evolutionary Genetic Analysis 5 (MEGA5; Tamura et al., 2011) program using the Maximum Likelihood method (10,000 bootstraps, Jones–Taylor–Thornton model). Finally the same alignment was also used to construct a NeighborNet Network (Figure S2 in Supplementary Material) with SplitsTree4 software (Huson and Bryant, 2006).

Based on these phylogenetic analyses, newly identified ZmPIN genes are named according to the cluster of the Arabidopsis and rice PIN family to which they belong.

Exon/intron structures of maize PIN genes were obtained from the MaizeGDB Genome browser¹¹, except for ZmPIN2 and ZmPINX that are not present in the genome database and for which exon/intron profiles have been drawn with GSDS, Gene Structure Display Server (Guo et al., 2007)¹² based on the alignment between the previously amplified and sequenced genomic and cDNA sequences.

ZmPIN map positions were identified by searching the www.maizesequence.org database and are manually depicted in Figure 2 and Figure S4 in Supplementary Material; the exact physical position is instead reported in Table S3 in Supplementary Material.

Duplication analysis of maize PIN genes and synteny between maize and rice chromosomes were identified and displayed by Synteny Mapping and Analysis Program (SyMAP) v3.4 (Soderlund et al., 2011)¹³

Finally, ZmPIN proteins transmembrane helices were predicted using TMHMM2 (Krogh et al., 2001)¹⁴: the red peaks in Figure 3 show the predicted transmembrane domains of proteins.

Zea mays B73 line was used for DNA and RNA extraction, for expression analysis, immunolocalization, and in situ hybridization. Plants were grown in the field or greenhouse, with supplemental lighting when necessary during winter/spring. Kernels were harvested and analyzed at different days after pollination (DAP) after manual self-pollination of plants in the field trials. Roots were collected 2–3 days after kernel germination in rolled adsorbent paper towels soaked with water in a growth chamber at 26°C with a photoperiod of 16 h light and 8 h dark as described in Woll et al., 2005. Kernels were previously surface sterilized with 70% ethanol for 10 min and then rinsed three times with water.

The maize mutant line br2 (Multani et al., 2003; Pilu et al., 2007) was kindly supplied by Dr. Roberto Pilu (Università di Milano) after introgression for six generations in B73 background. Homozygous br2 mutant plants were grown as previously described. Furthermore, the reporter lines pZmPIN1a::ZmPIN1a:YFP and DR5::mRFP (Gallavotti et al., 2008)¹⁵, kindly provided by Prof. Dave Jackson, were introgressed for three additional generations in B73 keeping the transgene in a hemizygous state.

Genomic DNA was extracted from maize leaves with the DNeasy Plant Mini Kit (QiAgen) according to the manufacturer’s instructions. Total RNA was extracted from maize tissues (seedlings, roots, leaves, tassels, ears, and kernels) using to the RNeasy Plant Mini Kit (QiAgen) and subjected to on-column DNase treatment (QiAgen). Before retrotranscription, total RNA was quantified by spectrophotometer analysis.

For the semi-quantitative RT-PCR expression analysis, ZmPIN and ZmABCB1/BR2 gene transcriptions were investigated in vegetative and reproductive tissues and kernels. Vegetative tissues included 3 days-old seedlings, young leaves, the apex (from 0 to 1.5 cm) and the elongation/mature zone (from 1.5 to 3 cm) of primary roots, and the seventh and fifth node, while for the reproductive tissues ear and tassel at the stage of 1 and 3 cm were considered. Kernels collected at different DAP (0, 3, 5, 6, 7, 8, 9, 10, 12) were also taken into account for the ZmPIN expression analysis.

For the detailed quantitative PINs expression analysis in B73 line and br2 mutant, young roots were collected from seedlings vertically grown in a growth chamber for 3 days as previously described. These roots were divided into specific segments, which were labeled from A to E indicating the segment from 0 to 0.2 cm (A), from 0.2 to 0.5 cm (B), from 0.5 to 1 cm (C), from 1 to 1.5 cm (D), and from 1.5 to 2 cm (E; Figure 6A). In these root segments the expression of ZmPIN genes and ZmABCB1/BR2 was investigated by Real-Time PCR.

For both expression analyses, cDNA synthesis was performed with the SuperScript III reverse transcriptase kit (Invitrogen), according to the manufacturer’s instructions. One microgram of total RNA was used as a template together with 1 μl oligo (dT) 12–18 (0.5 μg/μl – Invitrogen). In the semi-quantitative RT-PCR expression analysis, each cDNA was then diluted 1:10 and 1 μl was used for PCR amplification in a volume of 25 μl. The constitutively expressed GAPC2 gene was used as housekeeping internal control of the cDNA/RNA quantity. Expression analyses were performed with at least two primer combinations for each gene. Primer sequences are reported in Table S2 in Supplementary Material. In order to obtain semi-quantitative results, the number of cycles of the PCR was adjusted for each gene to obtain barely visible bands in agarose gels (see Table S2 in Supplementary Material for the number of amplification cycles for each gene). Aliquots of the PCR were loaded on 1.2% agarose gels and stained with Sybr-Safe (Invitrogen). For all PINs the different primer combinations tested gave comparable amplification results. ZmABCB1/BR2 amplification products (see Pilu et al., 2007 for primers used in RT-PCR amplification) were checked on 2% agarose gel. Images were captured with Gel Logic 100 Imaging System and Molecular Imaging software (Kodak).

Quantitative Real-Time PCR expression analysis was performed using an ABI 7500 Real-Time PCR System (Applied Biosystems) and the POWER SYBR^® GREEN PCR Master Mix (Applied Biosystems), following the manufacturer’s guidelines. Real-time conditions were: 2 min at 50°C, 10 min at 95°C, 40 cycles of: 15 s at 95°C and 1 min at 60°C. For each reaction, we observed product melting curves by heating from 60 to 95°C at 0.2°C/s. For all transcripts, this procedure allowed identification of a single product, which we confirmed by analysis on 2% agarose gel. Two different biological samples, obtained by different RNA preparations from separate root pools, were processed; three replicates were carried out for each primer combination and for each biological sample. The absolute quantification was carried out by the generation of standard curves for each gene. Expression levels of each auxin transporter gene were normalized to GAPC2 transcript quantities. Primer sequences are reported in Table S2 in Supplementary Material.

In situ hybridization experiments were conducted as described by Varotto et al., 2003. Plant material (kernels, vegetative apexes, roots, and inflorescences) was fixed in 4% paraformaldehyde (Sigma) in 0.1 M phosphate buffer (pH 7.2) for 16 h at 4°C, then dehydrated by ethanol series and embedded in Paraplast Plus (Sigma). Sections (7–10 μm) were cut using a Leica RM 2135 microtome (Leica, Germany) and collected on xylane-coated SuperFrost Plus slides (Menzel-Glazer). Slides were then deparaffinized and treated with 10 μg/ml proteinase K.

In vitro transcription of the DIG-UTP (Roche) labeled RNA sense and antisense probes was obtained using T7 and SP6 polymerases. More information on primer sequences used to amplify the probes for each PIN gene can be found in Table S2 in Supplementary Material. The hybridization was performed in a 50% formamide buffer at 48°C overnight. DIG detection and signal visualization were done using Anti-Digoxigenin-AP antibody (Roche) and NBT plus BCIP (Roche), following the manufacturer’s instructions. Slides were air-dried and mounted with DPX mounting medium (Fluka Biochemika). Negative controls obtained with sense riboprobes are reported in Figures S6A–D in Supplementary Material.

Zea mays B73 line and br2 mutant seeds were surface sterilized with 70% ethanol for 10 min and then rinsed three times with sterile water. Kernels were placed on soaked 3MM paper in horizontal position for 1 day. After imbibition, the seeds were transferred in rolled adsorbent paper towels in vertical position for successive treatments. Seedlings were treated with 50 μM NPA (1-naphthylphthalamic acid, a PAT inhibitor), 50 μM NAA (2-naphthalen-1-ylacetic acid, a synthetic auxin), and water as control growth condition for 2 days. Roots were collected and divided in two specific segments indicated with A (from 0 to 0.2 cm) and B (from 0.2 to 0.5 cm).

The expression profiles of ZmPIN genes and ZmABCB1/BR2 in untreated, NPA- and NAA-treated roots were investigated by Real-Time PCR using the standard curve method, as reported above.

The reporter lines pZmPIN1a::ZmPIN1a:YFP and DR5::mRFP were treated with NPA and NAA at the same concentrations and observed at the confocal microscope.

PIN1 immunostaining was performed on wax-embedded (9:1 – PEG400 distearate:1-hexadecanol 99%, Sigma) roots collected on 2–3 days-old plants vertically grown on rolled paper. Roots were fixed in 4% paraformaldehyde in PBS 1× solution for 1 h under vacuum conditions at room temperature, embedded, and cut (10–12 μm) with a Leica RM 2135 microtome (Leica, Germany). After dewaxing sections were blocked for 2 h in 1% BSA in 1× PBS buffer and stained overnight with the anti-AtPIN1 antibody (aP20 sc-27163, Santa Cruz Biotechnology) at a 1:250 dilution in PBS 1× and 1% BSA. The next day, the sections were washed twice for 10 min in PBS 1× and stained for 1 h with the secondary antibody (anti-goat IgG conjugated with Alexa568, Molecular Probes) diluted 1:400 in PBS 1×. Sections were washed twice for 10 min with PBS 1× and mounted with VECTASHIELD^® with DAPI Mounting Medium (Vector Laboratories, USA). For the negative controls of immunostaining experiments hybridization was performed with the secondary antibody only.

A monoclonal anti-IAA antibody (Biofords – Agdia – PMD09346/0096) was used for IAA immunolocalization in maize roots. Tissues were prefixed in 3% (w/v) N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC, Sigma) 0.1 M in PBS 1×, post-fixed in 4% paraformaldehyde in the same buffer and embedded in Paraplast Plus (Sigma). Sections (7–10 μm) were cut using a Leica RM 2135 microtome (Leica, Germany), collected on polysine slides (Menzel-Glazer) and processed as previously reported by Avsian-Kretchmer et al., 2002. The anti-IAA primary antibody was used at a concentration of 0.05 mg/ml. The secondary antibody, an anti-mouse IgG-AP (Santa Cruz Biotechnology), was used at a 1:250 dilution. Signal visualization was done using SIGMAFAST™ Fast Red Tablets (Sigma) and when purple was observed, the colorimetric reaction was stopped in water. Negative controls of IAA immunostaining experiments were performed on samples no pre-fixed with EDAC or hybridizing slides with the IgG-AP secondary antibody only and are reported in Figures S6E–G in Supplementary Material.

pZmPIN1a::ZmPIN1a:YFP and DR5::mRFP reporter lines roots were hand sectioned, mounted with 50% glycerol in water and observed with a Leica TCS SP2 laser confocal microscope (Leica Microsystems, Heidelberg, Germany) using the follow settings: (i) ZmPIN1a–YFP excitation at 514 nm and signal collection between 530 and 580 nm; (ii) DR5 excitation at 543 nm and signal collection between 580 and 640 nm. Images were coded red for RFP and yellow for the YPF reporter line.

Immunolocalization and in situ hybridization images were taken with a Leica DM4000B Digital microscope, equipped with a Leica DC300F Camera, and Leica Image Manager 50 software (Leica Microsystems, England). Images were coded red for Alexa568 and blue for DAPI.

Members of the maize PIN family were initially identified by a comprehensive Blast search in public databases using A. thaliana and O. sativa PIN transcript and protein sequences as queries. MaizeGDB and MaizeSequence were used as primary databases to obtain the genomic sequences coming from the B73 Maize Genome Sequencing project, release 5b.60, and their relative annotations, while GSSs from PlantGDB and from J. Craig Venter Institute, transcript sequences from NCBI and TAs from DFCI/TIGR were used to initially confirm and integrate the previously identified sequences and splicing models. In addition to the three ZmPIN1 genes previously characterized (Carraro et al., 2006; Forestan et al., 2010), nine full-length sequences were found in the genomic release 5b.60 and a tenth sequence was identified in a GSS from PlantGDB. Furthermore, two partial, not overlapping, GSSs were assembled together using a sorghum PIN sequence to fill the gap, for a total of 11 new putative auxin efflux carrier genes. A preliminary in silico characterization was performed to identify putative ORFs and amino acid sequences. All the full-length sequences were later amplified from genomic DNA and cDNA (except ZmPIN5b and ZmPIN10b for which the amplification of the cDNA full-length proved unsuccessful), to validate the sequences of the 11 maize PINs that were named according to the cluster of Arabidopsis and rice PIN family to which they belonged.

The phylogenetic relationships between early-land plants, monocots and eudicots PIN proteins were investigated including the new maize PIN proteins in a phylogenetic analysis (Figure 1; Figures S2 and S3 in Supplementary Material), together with PIN proteins of A. thaliana, P. trichocarpa, O. sativa, B. distachyon, P. patens, and S. moellendorffii. A wider evolutionary study was performed by also including PIN proteins of M. truncatula, V. vinifera, S. bicolor, and S. italica (Figure S1 in Supplementary Material). PIN protein sequences of different species were retrieved from Phytozome v7.0 and analyzed with different sequence alignment and phylogenetic analysis tools, to develop a robust evolutionary model of transporters. Auxin efflux carrier family members were aligned with ClustalX 1.81 (Blosum Weight Matrix; Gap Opening Penalty: 5; Gap Extension Penalty: 0.20; Thompson et al., 2002) and a neighbor-joining phylogenetic tree was prepared with the Phylip package (Felsenstein, 1989) using 100 bootstraps (Figure 1; Figure S1 in Supplementary Material). In addition, PIN proteins were aligned with MAFFT algorithm (Katoh et al., 2002, 2009) and phylogeny inferred using MEGA5 program (Maximum Likelihood method; 10,000 bootstraps; Jones–Taylor–Thornton model; Tamura et al., 2011; Figure S3 in Supplementary Material) or constructing a NeighborNet Network with SplitsTree4 software (Huson and Bryant, 2006; Figure S2 in Supplementary Material). The three different approaches showed comparable results on the evolutionary history of auxin efflux carrier proteins in early-land plants, monocots and dicots, also suggesting the evolutionary relationships between the 11 newly identified maize proteins, Arabidopsis, and rice PIN proteins (Figure 1; Figures S1–S3 in Supplementary Material). Indeed, the four different phylogenetic trees show the same clustering of newly identified maize PIN proteins with Arabidopsis and rice orthologs, while differences in few nodes with low support can be observed for V. vinifera and P. trichocarpa proteins (Figure 1; Figures S1–S3 in Supplementary Material).

In addition to the previously characterized ZmPIN1a, ZmPIN1b, and ZmPIN1c, a fourth gene phylogenetically close to AtPIN1 was identified. We named this gene, which clusterizes together with rice OsPIN1c and OsPIN1d genes, ZmPIN1d. The PlantGDB database search and the phylogenetic analysis also allowed identification of a putative ZmPIN2 gene; indeed the two available partial GSSs codify for a protein closely related to AtPIN2 and OsPIN2.

The gene structure analysis showed that also the fourth ZmPIN1 gene, ZmPIN1d, has six exons, whose distribution is similar to that of AtPIN1 and the four OsPIN1 genes (Figure 2A). Like the Arabidopsis and rice orthologs, ZmPIN2 gene presents a complex exon/intron structure, with seven exons and six introns. We also identified four genes that encode for proteins lacking the central hydrophilic domain that normally characterizes long PIN proteins. This peculiar hydropathic profile is typical of the Arabidopsis PIN5 and PIN8 proteins and our phylogenetic analysis confirmed that three of the maize proteins clusterize with Arabidopsis and rice PIN5 (ZmPIN5a, ZmPIN5b, and ZmPIN5c), while the remaining one represents the maize PIN8 ortholog (ZmPIN8). So, as recently reported in rice, three duplicated PIN5 genes and a PIN8 gene are also present in the maize genome. ZmPIN5a and ZmPIN5b have four introns, while ZmPIN5c has three with the first of about 2 kb in length (Figure 2A). As described in the Section “Material and Methods,” genome annotation of PIN5b gene showed the presence of a stop codon soon after the first splicing site, resulting in a truncated protein (Figure 2A). Anyway, the alignment with the sorghum ortholog CDS showed that the splicing model annotated could be wrong and a shift of a single base pair resulted in a full-length PIN5 protein. Unfortunately RT-PCR using several primer combinations proved unsuccessful in the amplification of the complete CDS. Moreover no ESTs or TAs corresponding to ZmPIN5b were found on public databases and a recent Genome-Wide Expression Atlas revealed that ZmPIN5b is not expressed in the 60 distinct analyzed tissues representing 11 major organ systems of inbred line B73 (Figure S5 in Supplementary Material; see text footnote 1; Sekhon et al., 2011). ZmPIN8 has a very similar exon/intron structure to that of OsPIN8 (Figure 2A).

Finally, we identified three proteins that do not clusterize with any Arabidopsis PIN proteins, confirming, as suggested for rice, the existence of monocot-specific PIN genes. Based on the phylogenetic analysis, these maize genes were named ZmPIN9, ZmPIN10a, and ZmPIN10b. These monocot-specific PIN genes have a conserved structure when compared to their rice counterparts. Phylogenetic analyses also revealed that ZmPINX and ZmPINY proteins clusterize together with the Arabidopsis PIN-like proteins (Figure S1 in Supplementary Material).

Chromosome map positions of maize PINs have been identified by genome browser searches and are reported in Figure 2B and Figure S4 in Supplementary Material. Duplication analysis revealed that ZmPIN1a, ZmPIN1b, and ZmPIN1c are located in duplicated blocks on chromosomes 9, 5, and 6, respectively (Figure 2C; Figure S4 in Supplementary Material). Interestingly, both maize and rice PIN5a, PIN8, PIN9, and PIN10a map on highly syntenic regions of chromosomes 3 and 1, respectively (Figure S4 in Supplementary Material, Wang et al., 2009).

The predicted transmembrane architecture of the ZmPIN proteins showed the typical structure with a conserved N-terminal region of transmembrane motifs, a variable middle domain representing the cytoplasmic domain of transmembrane and a C-terminal domain of transmembrane segments (Figure 3). Interestingly, ZmPIN9 putative protein presents a hydropathic profile in between that of long PINs (AtPIN1 and AtPIN2, which are plasma membrane localized) and that of short PINs (the ER-localized AtPIN5 and AtPIN8).

The tissue-specific expression pattern of 12 ZmPIN genes (ZmPIN1a, ZmPIN1b, ZmPIN1c, ZmPIN1d, ZmPIN2, ZmPIN5a, ZmPIN5b, ZmPIN5c, ZmPIN8, ZmPIN9, ZmPIN10a, and ZmPIN10b), two ZmPIN-like genes (ZmPINX and ZmPINY) and BR2, a maize MDR/PGP/ABCB efflux transporter were analyzed by semi-quantitative RT-PCR and the results are reported in Figure 4. These results are in agreement with those obtained by the genome-wide transcriptome analysis previously mentioned and reported in Figure S5 in Supplementary Material (Sekhon et al., 2011).

ZmPIN1a, ZmPIN1b, and ZmPIN1c expression analysis confirmed that these genes are ubiquitously expressed but differentially modulated in maize vegetative and reproductive tissues and during kernel development (see also Carraro et al., 2006; Forestan et al., 2010). A different expression pattern was observed for ZmPIN1d that is specifically expressed in tassels, ears and in the fifth node of adult plants and not detectable in caryopses after pollination. ZmPIN2 is expressed in root tip, male and female inflorescences and its expression is modulated during the early stages of kernel development. ZmPIN5a is mainly expressed in the elongation/mature zone of the primary root, in nodes and in the young seed till 10 DAP. ZmPIN5b is up-regulated in the fifth node, and has a weak expression in male and female inflorescences and during kernel development (note that the amplified fragment corresponds to the first exon while no amplifications were obtained with downstream located primers). ZmPIN5c is highly expressed during kernel development from 3 to 12 DAP. ZmPIN8 is expressed in all the tissues analyzed with the exception of the root and is detectable during kernel development, where it appeared up-regulated between 6 and 9 DAP. Of the monocot-specific genes, ZmPIN9 is exclusively expressed in roots and nodes while ZmPIN10a is expressed during male and female inflorescence development and at very low level during kernel development. Finally a very weak expression of ZmPIN10b is detectable in young ears and tassels.

The expression analyses of the two maize PIN-like genes showed that they are ubiquitously expressed and differentially up-regulated in all the analyzed tissues. In detail, ZmPINX is up-regulated in root apex and male and female inflorescences, while ZmPINY is highly expressed during kernel development.

Finally, BR2 transcript is detectable in all the tested tissues with a lower expression level in the root elongation zone compared to other vegetative and reproductive tissues. Moreover, the BR2 transcript amount is detectable at a very low level during tested kernel development stages.

The longitudinal structure of the maize root includes the root cap at the terminal end, the root apical meristem, and the elongation and root hair zones. In cross section, the root is organized in a series of concentric cell layers: the outermost epidermic layer and exodermis, the pluristratified cortex, the endodermis, and the pericycle that surrounds the central vascular cylinder (see Hochholdinger et al., 2004 for a review on root development in maize). Six ZmPIN genes are expressed in maize root: indeed, quantitative RT-PCR data showed that ZmPIN1a, ZmPIN1b, and ZmPIN1c and the monocot-specific ZmPIN9 are expressed both in root apical and distal zones (Figure 4). In addition, ZmPIN2 appeared up-regulated in root apex, while ZmPIN5a is up-regulated in the root elongation/mature zone (Figure 4). The expression pattern of ZmPIN genes expressed in the root apex was determined at tissue level using in situ hybridization (Figures 5A–O). In root apex longitudinal sections, comprising the root cap and meristematic area, a ZmPIN1 probe, which recognized the three gene transcripts expressed in the root showed the hybridization signal in the root cap with a clear up-regulation in calyptrogen, in the root meristematic area, in the epidermis and in the differentiating vascular tissues of the central cylinder (data not shown). Specific probes for ZmPIN1a, ZmPIN1b, and ZmPIN1c were also transcribed in vitro DIG-labeled and hybridized on root apex longitudinal sections. The results showed that ZmPIN1a is the only PIN1 transcript localized in the calyptrogen and in the meristem region of the root (Figure 5A), while ZmPIN1b is mainly expressed in epidermis, root cap, and vasculature (Figures 5B–D). Finally a specific ZmPIN1c probe showed that ZmPIN1c is localized in the epidermis and vasculature of the central cylinder (Figures 5E–G).

The localization pattern of the three ZmPIN1 genes was also confirmed by immunolocalization experiments, using an anti-AtPIN1 antibody, which was shown to recognize the proteins of the three maize PIN1 genes expressed in the root. PIN1 proteins are polarly localized in the epidermis, meristem region, and central cylinder, where they are inserted in the basal cell membrane (Figure 5P) while they are detectable in the cytoplasm of the calyptrogens and proliferating cap (Figure 5Q). At protein level, the localization pattern of ZmPIN1a protein was observed using a ZmPIN1a–YFP reporter line (Gallavotti et al., 2008). At the confocal microscope the YFP signal was evident in the basal cell membranes of the central cylinder cells and in the cytoplasm of root cap cells and meristematic region of the root apex (Figures 5T–V and 7H).

ZmPIN2 transcripts were localized in root epidermis, exodermis, and multiple layers of cortex in cross section of the root apex (Figures 5H–M). Finally, ZmPIN9 the monocot-specific PIN gene is mainly expressed in endodermis, pericycle, and in phloem of the central cylinder (Figures 5N,O).

To correlate the localization pattern of the PIN genes expressed in maize root with auxin distribution and maximum in the same tissues, observations at the confocal microscope of a DR5::mRFP reporter line (Gallavotti et al., 2008) and immunolocalizations with an anti-IAA antibody on root longitudinal sections were both performed. Auxin response was observed in the root cap and vasculature in the DR5-RFP reporter line fresh root dissected at the confocal microscope (Figures 5W–Y and 7A,B). Auxin accumulation was evident in the root cap, in particular in the calyptrogen, in the epidermis at the level of root meristem and vasculature in longitudinal root sections labeled with the anti-IAA antibody (Figures 5R–S).

The br2 maize mutant exhibits reduced shootward auxin transport at the root apex and reduced root gravitropic growth (McLamore et al., 2010). To investigate whether this alteration in auxin fluxes influenced the expression of ZmPIN transporters, the mRNA levels of ZmPIN1a, ZmPIN1b, ZmPIN1c, ZmPIN2, and ZmPIN9 genes, the PIN members expressed in root apex, were analyzed by Real-Time PCR in different segments of B73 and br2 young primary roots.

As shown in Figure 6B, in br2 mutant the expression level of ZmABCB1/BR2 transcript was drastically reduced in all root segments (from A to E) compared to the levels observed in B73 line. ZmPIN1a transcript showed a slight higher expression in the root segments A–C and lower levels in segments D–E of br2 mutant when compared with the same segments of B73. On the contrary, the mRNA level of ZmPIN1b was higher in A–B–C root segments of br2 mutant, while in the D–E segments its level was comparable to that observed in wt. Also ZmPIN1c showed a different expression pattern in br2 compared to B73 line: in particular, an up-regulation was observed in segment A, while a down-regulation occurs in segment D.

The transcript level of ZmPIN2 was slightly higher in all tested segment of br2 mutant, in particular in the distal region of the root (segments D and E) where PIN2 is not detectable in B73. Finally, ZmPIN9 transcript was up-regulated in segments C–D of br2 mutant.

Taken all together these results highlight that the expression profile of PIN transporters is altered in br2 mutant primary roots.

The expression profile of ZmPIN genes and ZmABCB1/BR2 was investigated by Real-Time PCR in root apex of B73 and br2 mutant line grown in water (control), in presence of exogenous auxin (NAA), and of a PAT inhibitor (NPA; Figure 6C). Due to the strong reduction of root elongation caused by NAA application, the analysis was done just on the first two root segments indicated with A (from 0 to 0.2 cm) and B (from 0.2 to 0.5 cm).

In both lines the addition of NAA and NPA affected the expression of ZmPIN genes and ZmABCB1/BR2 in both root segments. In particular in B73, NPA strongly inhibited ZmABCB1/BR2 expression in segment A, while NAA application doubled its expression levels in segment B. The same NAA effect was observed in br2 mutant roots. Both NAA and NPA treatments increased ZmPINa and ZmPIN1c transcript levels in segment B in both genotypes; on the contrary NAA application strongly reduced ZmPIN1c levels in the segment A of br2 root. Similarly, ZmPIN1b transcript levels were strongly reduced after NAA application in br2 mutant roots (segments A and B), while NPA affected its expression exclusively in the B segment. NAA treatment increased ZmPIN9 transcript levels in the segment B of mutant roots, while ZmPIN2 expression was affected in a lower extent by the drug applications.

Experimental evidences showed that auxin transporter expression is altered by exogenous auxin or PAT inhibitor applications and these alterations are more dramatic in the br2 mutant background.

The effects of NAA and NPA applications were investigated at cellular level by microscope observation of DR5::mRFP and ZmPIN1a–YFP reporter lines (Figure 7). NAA applications caused the main effects on root anatomy, auxin responses, and PIN1a localization (Figures 7B,E,F,I,K). Exogenous auxin application indeed resulted in a size reduction of both root cap and meristem (compare Figures 7B,D) and in the formation of a pluristratified epidermis (Figures 7E,F). After the treatment, DR5 activity is diffused in the lateral root cap cells (Figure 7B) and not limited to the root cap tip as in the control (Figure 7D). Auxin response was detectable in the vasculature that differentiates very close to the root meristem (Figures 7B,E) and also the pluristratified epidermis showed DR5 activity (Figure 7F). Although PIN1a–YFP always localized in the stele cells in both NAA-treated and control roots, some observed differences in protein localization reflected the changes in root morphology, particularly at the level of the vasculature and meristem (Figures 7H,I,K).

Following NPA applications, auxin signal was detectable in root cap as in the control (data not shown) while a diffuse DR5 activity marked the whole central cylinder and the cortex cells (Figures 7C,G). This suggests that NPA caused a failure in creating the auxin maximum detectable in the vascular cell in the control roots (Figure 7A). Similarly, the ZmPIN1a–YFP reporter line showed a more diffuse localization of the PIN1a protein that marked also the cortex cells (Figures 7J,L).

In maize, at the transition from vegetative to reproductive development the shoot apical meristem (SAM) becomes an inflorescence meristem (IM) which starts to produce lateral meristems called branch meristems (BMs). BMs develop spikelet-pair meristems (SPMs), each bearing two spikelet meristems (SMs). SMs form two floral meristems (FMs), the upper FM and lower FM, which produce the floral organ of the tassel. In each flower of the tassel, after gynoecial development, the gynoecium degenerates, and an imperfect staminate flower results. Within a few weeks after the SAM transition, a lateral shoot meristem in the axil of a leaf becomes an ear IM and produces multiple rows of SPMs, which follow the same step as in the tassel until the lower floret aborts as do the stamens of the upper floret. Indeed, in the mature ear a pistillate flower is present per spikelet (see McSteen et al., 2000 for a review on maize inflorescence development). Since ZmPIN1d transcript is specifically up-regulated in both male and female inflorescences, in situ hybridizations were performed in the SAM, starting from the transition stage to SPMs and spikelet differentiation, in male inflorescences, and during female inflorescence development (Figures 8A–H). During flowering transition, which is characterized by the elongation of the SAM and the adoption of an inflorescence identity to form a tassel primordium, ZmPIN1d hybridization signal is detectable in the L1 layer of the IM apical meristem (Figure 8A); later on, ZmPIN1d is localized in the L1 layer of the developing tassel SPMs primordium (data not shown). In the ear, ZmPIN1d showed the same localization patterns as in the tassel, being localized in the L1 layer of the IM during the transition phase and maintaining its localization in the L1 cell layer during SPM differentiation (Figures 8B,C). During SMs differentiation and growth, ZmPIN1d is localized in the tips of the developing glumes (Figure 8D). Later on in ear development, during the pistillate flower differentiation, ZmPIN1d hybridization signal is detectable in the outer cell layers of the gynoecium, till the differentiation of the gynoecium ridge that will encircle the ovule region giving rise to the stylar canal (Figures 8E–G) and in the nucellus surrounding the mother cells of the megaspore (Figure 8H). The same localization pattern was observed in the pistillate flowers of female inflorescence sections hybridized with ZmPIN1a (Figures 8I–K), ZmPIN1b (Figure 8L), and ZmPIN2 (Figures 8M,N) antisense DIG-labeled mRNA probes.

RT-PCR showed that ZmPIN2, ZmPIN5c, ZmPIN8, and ZmPIN10a are up-regulated during the early phases of kernel development, from 3 to 12 DAP. To precisely determine the localization of these genes at tissue level, in situ hybridization experiments were done on longitudinal sections of maize kernels from 5 to 12 DAP (Figure 9). During this early stage of maize kernel development the main cellular domains of the endosperm, the Basal Transfer Layer (BETL), the embryo surrounding region (ESR), the aleurone, and sub-aleurone differentiate, before the accumulation of reserves occurs. At 5 DAP ZmPIN2 is expressed in the differentiating endosperm ESR and in the embryo proper (Figure 9B); at 12 DAP later on during kernel development when the main cellular domains of the endosperm are completely differentiated, ZmPIN2 transcript is localized in the BETL, ESR, aleurone, and developing embryo at the transition stage (Figures 9A,C,K). ZmPIN5c transcripts are localized in ESR, BETL, and maternal chalazal cells of the kernel and in the embryo at both 5 and 12 DAP (Figures 9D–G); at 12 DAP its hybridization signal appears particularly evident in the outer epidermic layer of the embryo scutellum (Figure 9F), in the transition zone between BETL and aleurone (Figure 9G), while transcripts are not detectable in the aleurone layer (Figure 9L). ZmPIN10a transcript is also localized in both BETL and ESR endosperm cellular domains and embryo starting at 5 DAP (Figures 9H–I), and are also detectable in the chalazal and pedicel kernel tissues at 12 DAP (Figure 9H). Finally, ZmPIN8 is expressed in BETL cells, maternal chalazal tissues (Figure 9J), and aleurone layer (Figure 9M).