Seeds of the 21-3 inbred line of brachypodium (Vogel and Hill, 2008) were sterilized in 100% ethanol for 1 min and rinsed three times with sterile water. The lamella and palea were softened by further incubation in sterile water for 2 h and removed with forceps, taking care not to damage the seed. Prepared seeds were then spread onto rinsed perlite irrigated with a standard hydroponic solution, described below. After stratification at 4°C for 24 h, plants were grown for 7 days at 22°C; 12-h light/12-h dark photoperiod at photosynthetic photon flux density of 150 μmol photons m⁻²s⁻¹ before transferring to a hydroponic solution prepared as described (Arteca and Arteca, 2000) except that Cu (as CuSO₄) was added at a higher concentration of 0.25 μM. For Cu limitation and sensitivity assays, 7-day-old seedlings were transferred from perlite to hydroponic solution supplemented with the indicated concentrations of CuSO₄, or without CuSO₄ but with the specific Cu chelator, bathocuproine disulfonate (BCS) (Rapisarda et al., 2002). Plants were grown for 18 days before subsequent analyses. For all experiments, the hydroponic solution was changed every 7 days.

Root and leaf tissues were separated from 25-day-old plants grown under the indicated conditions and flash-frozen in liquid nitrogen. Samples were homogenized in liquid nitrogen using a mortar and a pestle, and total RNA was isolated using the Plant RNA Kit (Omega Bio-Tek), according to the manufacturer's instructions. Genomic DNA in RNA samples was digested with DNAse I (Omega Bio-Tek) prior to cDNA synthesis using the iScript cDNA Synthesis kit (BioRad).

Prior to qRT-PCR analysis, primers (Supplemental Table 1) and cDNA concentrations were optimized to reach a qRT-PCR amplification efficiency of 100 ± 10%. Two microliters of 10-fold diluted cDNA was used as a template in a total reaction volume of 10 μl containing 500 nM of each PCR primer, 50 mM KCl, 20 mM Tris-HCI, pH 8.4, 0.2 mM dNTPs, and 1.25 units of iTaq DNA polymerase in iQ SYBR Green Supermix (BioRad), containing 3 mM MgCl₂, SYBR Green I, 20 nM fluoresceine, and stabilizers. PCR was carried out using the CFX96 Real-Time PCR system (BioRad). The thermal cycling parameters were as follows: denaturation at 95°C for 3 min, followed by 39 cycles of 95°C for 10 s then 55°C for 30 s. Amplicon dissociation curves, i.e., melting curves, were recorded after cycle 39 by heating from 60 to 95°C with 0.5°C increments and an average ramp speed of 3.3°C s⁻¹. Data were analyzed using the CFX Manager Software, version 1.5 (BioRad). A brachypodium gene encoding ACTIN2 was used as a reference for normalizing gene expression. qRT-PCR experiments were conducted using three independent biological samples, each consisting of three technical replicates, unless indicated otherwise. Statistical analysis was performed using the Relative Expression Software Tool (REST, Qiagen; Pfaffl et al., 2002).

Total RNA isolated from brachypodium leaves and oligonucleotides pairs that are indicated in Supplemental Table 1 were used for RT-PCR amplification of BdCOPT1, BdCOPT3, BdCOPT4, and BdCOPT5 cDNAs with or without the stop codon. Primers were designed to contain attB sites on resulting PCR products for subsequent Gateway cloning (Invitrogen) into the DONR-Zeo entry vector (Invitrogen) and appropriate destination vectors described below.

S. cerevisiae SEY6210 (MATa ura3-52 leu2-3,-112 his3Δ200 trp1Δ901 lys2-801 suc2Δ9) wild-type and the ctr1Δctr2Δctr3Δ triple mutant (MATa ura3-52 his3Δ200 trp1-901 ctr1::ura3::Knr ctr2::HIS3 ctr3::TRP1) used for functional complementation assays were the generous gift of Dr. Dennis Thiele (Duke University). Yeast cells were transformed with YES3-Gate-BdCOPT1, YES3-Gate-BdCOPT3, YES3-Gate-BdCOPT4, YES3-Gate-BdCOPT5 constructs or an empty YES3-Gate vector using the Frozen-EZ yeast Transformation II Kit (Zymo Research). Transformants were selected for uracil prototrophy on YNB medium (YNB-Ura) containing 0.67% (w/v) Yeast Nitrogen Base without amino acids (Difco), 0.077% (w/v) CSM-Ura, 0.05% (w/v) NaCl, 2% dextrose, 2% (w/v) agar. Respiration competence was evaluated by testing the ability of transformants to grow on the non-fermentable carbon sources, glycerol and ethanol (YPEG) as described (Dancis et al., 1994a; Puig et al., 2002). Briefly, transformants were grown in liquid YNB-Ura to an OD_{600 nm} = 1.0, serially-10-fold diluted and spotted onto YPEG medium containing 1% (w/v) yeast extract, 2% (w/v) bacto-peptone, 3% (v/v) glycerol 2% (v/v) ethanol, and 2% (w/v) agar and the indicated concentration of CuSO₄ or onto YNB-Ura for controls. Plates were incubated for 3 days at 30°C.

Protoplasts were isolated from leaves of 25-day-old brachypodium grown as described above. The protoplast isolation procedure was based on (Zhai et al., 2009). Briefly, 0.2 g of young leaf tissue was immersed in 15 ml of filter-sterilized TVL solution (Supplemental Table 2), finely chopped with a fresh razor blade and transferred to a 200 ml beaker containing 20 ml of Enzyme solution (Supplemental Table 2). The beaker was wrapped in aluminum foil to protect samples from light and samples were vacuum infiltrated for 30 min before incubation at 30°C for 60 min. The mixture was then agitated at 35 rpm at room temperature for 18–20 h. Released protoplasts were collected into 50-ml Falcon centrifuge tubes by carefully sieving the mixture through eight layers of cheesecloth, pre-wetted with W5 solution (Supplemental Table 2). To increase the protoplast yield, the cheesecloth was rinsed with an additional 10 ml of W5 solution. Sieved protoplasts were then carefully overlaid with 5 ml of W5 solution and left at room temperature for 1 h to allow protoplasts to float to the interface of Enzyme solution and W5 solution. Fifteen milliliters of protoplasts were collected from the interface and transferred into a new 50 ml Falcon tube containing 20 ml of W5 solution. Protoplasts were collected by centrifugation for 7 min at 100 × g. The residual Enzyme solution was removed by two rounds of rinsing protoplasts with 10 ml W5 solution and centrifuged for 5 min at 60 × g. Purified protoplasts were resuspended in 3–5 ml W5 solution and the protoplast yield was evaluated by cell counting using a hemocytometer. Protoplast viability was evaluated using the Plant Cell Viability Assay Kit (Sigma), according to manufacturer's recommendations.

Protoplasts were transfected using a procedure adopted from (Jung et al., 2011). Briefly, purified protoplasts were incubated on ice for 30 min and centrifuged for 5 min at 60 × g and W5 solution was removed. Protoplasts were then re-suspended in MMG solution (Supplemental Table 2) and 100 μl aliquots were transferred into a 2-ml round-bottom microcentrifuge tube. Plasmid DNA (5–10 μg) was added to protoplasts and mixed gently. For controls, DNA was omitted and replaced with equivalent volumes of sterile water (mock transfection). Transfection was initiated by the addition of 110 μl of PEG-calcium solution (Supplemental Table 2). Protoplasts were gently mixed with PEG-calcium solution by tapping the tube followed by incubation for 7 min at room temperature. Transfection was terminated by diluting the mixture by an addition of 700 μl of W5 solution. Transfected protoplasts were collected by centrifugation for 2 min at 100 × g, and supernatant was removed to leave 50–100 μl of protoplast suspension. Each sample was then brought up to a volume of 1 ml with W5 solution and incubated in the dark at room temperature for 18 h.

For studies of the subcellular localization in brachypodium protoplasts, BdCOPT3, and BdCOPT4 cDNAs lacking the stop codon were fused at the C-terminus to the enhanced green fluorescent protein (EGFP) of the SAT6-N1-EGFP-Gate vector and expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The resulting 35S_pro-BdCOPT-EGFP constructs or SAT6-N1-EGFP-Gate lacking cDNA inserts were transfected into brachypodium protoplasts as described above. Plasma membranes were stained with 50 μM FM4-64 as described (Ueda et al., 2001).

For studies of subcellular localization in S. cerevisiae, entry clones containing BdCOPT1, BdCOPT3, BdCOPT4, or BdCOPT5 cDNAs without the stop codon were fused at C-terminus with EGFP in the YES3-EGFP-Gate vector (Jung et al., 2012). The resulting constructs and the empty YES3-EGFP-Gate vector were transformed into S. cerevisiae ctr1Δctr2Δctr3Δ triple mutant using the Frozen-EZ yeast Transformation II Kit (Zymo Research) and transformants were selected on YNB-Ura medium, as described above. Subcellular localization was assessed in cells grown overnight in liquid YNB-Ura.

EGFP- and FM4-64- mediated fluorescence, and chlorophyll autofluorescence were visualized using FITC (for EGFP) or rhodamine (FM4-64 and chlorophyll) filter sets of the Axio Imager M2 microscope equipped with the motorized Z-drive (Zeiss). Z-stack (1.3 μm-thick) images were collected with the high-resolution AxioCam MR Camera and then 3D deconvoluted using an inverse filter algorithm of the Zeiss AxioVision 4.8 software. Images were processed using the Adobe Photoshop software package, version 12.0.

Vectors and S. cerevisiae strains THY.AP4 (MATa leu2-3,112 ura3-52 trp1-289 lexA::HIS3 lexA::ADE2 lexA::lacZ) and THY.AP5 (MATα URA3 leu2-3,112 trp1-289 his3-Δ1 ade2Δ::loxP) for MYTH were obtained from the Frommer lab (Stanford University) depository at Arabidopsis Biological Resource Center (ABRC) http://www.arabidopsis.org/abrc/index.jsp. AtCOPT6-Cub-PLV and AtCOPT6-NubG fusions were previously described (Jung et al., 2012). Full-length BdCOPT1, BdCOPT3, BdCOPT4, and BdCOPT5 cDNAs without stop codons were introduced into the MetYC-dest and pXN-dest22-3HA destination vectors by Gateway cloning (Invitrogen) to generate bait, BdCOPT-CubPLV, and prey, BdCOPT-NubG, constructs in THY.AP4 and THY.AP5 strains, respectively. In all cases, C- and N terminal fragments of ubiquitin were placed at the C-terminus of the BdCOPT proteins. Protein interactions were selected in diploid cells after 2 days of growth on SC medium lacking adenine and histidine. Interactions were verified using β-galactosidase assays, as detailed in Kittanakom et al. (2009).

The phylogenetic tree was built using the Neighbor-Joining method (Saitou and Nei, 1987). The bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the number of differences method (Nei and Kumar, 2000) and are in units of the number of amino acid differences per sequence. The analysis involved 22 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 75 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011) with A. thaliana IRT1 as an outgroup.

Accession numbers for genes used in this study were according to nomenclature from ARAMEMNON 7.0 (http://aramemnon.botanik.uni-koeln.de/) and MIPS (http://mips.helmholtz-muenchen.de/plant/brachypodium/): Bradi1g24180 (BdCOPT1), Bradi1g24190 (BdCOPT2), Bradi2g51210 (BdCOPT3), Bradi4g31330 (BdCOPT4), Bradi5g09580 (BdCOPT5), Bradi1g10630.1 (BdACTIN), and At2g26975 (AtCOPT6).

To identify putative members of the CTR/COPT family in brachypodium, we used the Plant Membrane Protein database, ARAMEMNON 7.0 (http://aramemnon.botanik.uni-koeln.de/; Schwacke et al., 2003), which has the most complete annotation of putative CTR/COPT transporters based on amino acid similarity and motif organization of CTR/COPT proteins in different species. We found that the brachypodium genome possesses five genes that encode putative CTR/COPT transporters Bradi1g24180, Bradi1g24190, Bradi2g51210, Bradi4g31330, Bradi5g09580, designated COPT1 through COPT5 (alias BdCOPT1-BdCOPT5) (Table 1). BdCOPT1 and BdCOPT2 are located on chromosome 1, while BdCOPT3, BdCOPT4, and BdCOPT5 are found on chromosome 2, 4, and 5, respectively (Table 1). Based on the BdCOPT gene structures in the ARAMEMNON 7.0 database, BdCOPT1, BdCOPT3, BdCOPT4, and BdCOPT5 lack introns, similar to COPT genes in rice (Yuan et al., 2011) and A. thaliana (http://arabidopsis.org). In contrast, BdCOPT2 possesses two introns (Figure 1A).

BdCOPTs share 16–69% amino acid sequence identity and 26–73% sequence similarity to each other (Table 2). Phylogenetic analysis revealed that a majority of CTR/COPT proteins from brachypodium and O. sativa cluster together and are separate from CTR/COPTs from A. thaliana (Figure 1B). One exception is BdCOPT4, which forms a separate cluster along with AtCOPT5, OsCOPT7, and OsCOPT6 (Figure 1B). BdCOPT4 is highly similar (65%) to AtCOPT5, whereas other BdCOPT proteins share 38–54% similarity to their counterparts in A. thaliana (Table 3). Similar to OsCOPT members (Yuan et al., 2011), the nucleotide sequence of BdCOPT genes is GC-rich, ranging from GC content of 67.9% (BdCOPT2) to 73.2% (BdCOPT3), in contrast to an average GC content of 50% in A. thaliana COPTs.

CTR/COPT family members possess conserved structural features that include three putative transmembrane helices (TMs), the N- and C-termini located toward the extracellular space and cytosol respectively, N-terminally-located methionine-rich motifs (Mets motifs), and MXXXM-X₁₂-GXXXG motifs located within TM2 and TM3, respectively (Puig et al., 2002; De Feo et al., 2007, 2009; Peñarrubia et al., 2010). Importantly, MXXXM motifs of TM2 in Ctr1p of S. cerevisiae, and COPT2 and COPT6 of A. thaliana contain a positionally conserved Met residue that is essential for the ability of these proteins to transport Cu (Puig et al., 2002; Jung et al., 2012; Gayomba et al., 2013). Some CTR/COPT proteins also contain the C-terminal cysteine-rich CXC motif, which is suggested to bind Cu ions for transfer to cytosolic Cu chaperones, or to downregulate Ctr1p activity in response to toxic Cu levels (Puig et al., 2002; Xiao et al., 2004; Wu et al., 2009). This motif is present in A. thaliana COPT1 and COPT2, but is absent in COPT6; nevertheless, COPT6 is a functional Cu transporter (Jung et al., 2012).

Computer algorithm-assisted analysis of membrane topology and motif organization in brachypodium CTR/COPT proteins revealed that all BdCOPTs, except for BdCOPT3, are predicted to contain the classical three TMs (Figure 2) with N-terminal domains oriented toward the extracellular space, while C-terminal domains are predicted to be located in the cytosol. In contrast, BdCOPT3 is predicted to have two transmembrane domains, with both N- and C-termini located in the cytosol. While polypeptides of all BdCOPT included the N-terminal Mets motifs, the distribution of MXXXM-X₁₂-GXXXG motifs varied within different BdCOPT polypeptides (Figure 2). For example, MXXXM motifs and thus, positionally conserved essential Met residues, were located within TM2 in BdCOPT3, BdCOPT4, and BdCOPT5, but outside of TM2 in BdCOPT1 and BdCOPT2. Although the predicted membrane topology of BdCOPT proteins has to be validated experimentally, the location of essential positionally conserved Met residues of the MXXXM motif outside of the predicted membrane domain suggest that BdCOPT1 and BdCOPT2 might not mediate Cu transport. The C-terminal CXC motif is present in BdCOPT3 as a CC motif and in BdCOPT4 as a CXC motif, but is absent in BdCOPT1, BdCOPT2, and BdCOPT5 (Figure 2).

To determine whether BdCOPT genes respond transcriptionally to Cu status of the plant like their counterparts in A. thaliana and O. sativa, we first developed growth conditions under which Cu limitation would minimally affect the growth and development of brachypodium. Seeds were germinated in perlite irrigated with a modified hydroponic medium supplemented with 0.25 μM CuSO₄ (Supplemental Figure 1 and Methods and Materials) and grown for 7 days before transferring to hydroponic medium to grow further for 18 days under the following conditions: (1) control conditions (0.25 μM CuSO₄); (2) Cu limited conditions (0 μM CuSO₄); (3) Cu deficiency (0 μM CuSO₄ and supplemented with 500 μM of the Cu chelator, bathocuprione disulfonate (BCS); and (4) Cu excess (3 μM CuSO₄). While there were no signs of chlorosis in leaves of plants grown under Cu deficiency or Cu excess (Supplemental Figure 2A), plants grown under Cu deficiency had decreased height, and shoot and root dry weight compared to plants grown under control conditions (Supplemental Figure 2B). Plants grown under Cu excess had decreased shoot and root dry weight, but were of the same height as control plants. Cu limitation did not significantly alter plant growth or shoot and root biomass (Supplemental Figure 2B).

We then analyzed the steady-state levels of BdCOPT mRNAs in different plant tissues of brachypodium grown under the Cu regimes described above. Expression studies in roots (Figure 3A) revealed that Cu limitation increased mRNA expression of BdCOPT3 and BdCOPT4. Cu deficiency increased BdCOPT3 and BdCOPT4 expression even further and, in addition, increased expression of BdCOPT1. There were no statistically significant differences in mRNA expression levels of BdCOPT2 and BdCOPT5 in root tissue of plants grown under these conditions (Figure 3A). In roots of plants grown under Cu toxicity, BdCOPT4 was the only gene whose expression was responsive to this treatment (Figure 3A). In young leaves (Figure 3B), Cu limitation elevated the abundance of BdCOPT3 and BdCOPT4 transcripts, which increased even further under Cu deficiency. In young leaves of plants grown under Cu toxicity, expression of BdCOPT4 decreased, but expression of BdCOPT2 increased. Expression studies in old leaves (Figure 3C) of plants grown in Cu limited or Cu deficient conditions showed an increase of the abundance of BdCOPT3 and BdCOPT4 transcripts. In contrast, both, Cu toxic and Cu limited conditions, increased expression of BdCOPT1, while expression of BdCOPT2 and BdCOPT5 were downregulated under Cu deficiency.

To summarize, of five BdCOPT genes, BdCOPT3, and BdCOPT4, are the most responsive to fluctuations in Cu availability, and changes in their expression are most prominent in roots and young leaves. BdCOPT2 is significantly upregulated under Cu toxicity only in young leaves, while BdCOPT1 is regulated by Cu mainly in old leaves. Expression of BdCOPT5 was the least responsive to Cu availability and was downregulated only by Cu deficiency and only in old leaves.

We next tested whether BdCOPT proteins are involved in Cu transport. In this regard, the S. cerevisiae ctr1Δctr2Δctr3Δ mutant, which lacks high-affinity plasma membrane-localized Cu uptake transporters Ctr1p and Ctr3p, and the vacuolar membrane-localized Ctr2p, is deficient in Cu uptake and release of Cu from the vacuole, and has been successfully used to identify Cu transport capabilities of CTR/COPT transporters from higher plants (Kampfenkel et al., 1995; Barhoom et al., 2008; Yuan et al., 2010, 2011; Garcia-Molina et al., 2011; Klaumann et al., 2011; Jung et al., 2012; Gayomba et al., 2013). Analyses of Cu transport capabilities using yeast as a heterologous system is based on the fact that cells lacking functional Cu uptake systems are unable to deliver Cu to cytochrome c oxidase in the mitochondrial respiratory chain, preventing cell growth on non-fermentable carbon sources such as glycerol and ethanol (YPEG medium), unless high concentrations of exogenous Cu are added to the growth medium (Dancis et al., 1994b; Glerum et al., 1996). Therefore, we expected that if BdCOPT proteins act as high-affinity Cu transporters, then their expression in the S. cerevisiae ctr1Δctr2Δctr3Δ mutant would promote Cu uptake and suppress growth defects of the mutant on non-fermentable medium.

Here, we focused on analyses of BdCOPT1, BdCOPT3, BdCOPT4, and BdCOPT5 due to difficulty in cloning of BdCOPT2. As a positive control, we used the previously characterized CTR/COPT transporter from A. thaliana, AtCOPT6 (Jung et al., 2012). We found that all yeast lines grew at the same rate on standard medium (YNB-Ura) supplied with glucose as a carbon source or on YPEG medium supplemented with 100 μM CuSO₄ (Figure 4). In contrast, only the empty vector-expressing wild-type cells and ctr1Δctr2Δctr3Δ cells expressing AtCOPT6 grew well on YPEG medium (Figure 4), suggesting that none of BdCOPTs tested were able to rescue the growth defect of the S. cerevisiae mutant on YPEG medium. However, addition of a low concentration of Cu (10 μM CuSO₄) to YPEG medium allowed BdCOPT3 (Figure 4B), BdCOPT4 (Figure 4C), and BdCOPT5 (Figure 4D) but not BdCOPT1 (Figure 4A) to complement partially the growth defect of ctr1Δctr2Δctr3Δ cells. These results suggest that transport capabilities of brachypodium COPT transporters differ from their counterparts in A. thaliana.

S. cerevisiae Ctr1p and Ctr3p localize to the plasma membrane and contribute to Cu uptake into the cell (Dancis et al., 1994a; Peña et al., 2000), while Ctr2p localizes to the vacuolar membrane and remobilizes Cu from this internal store upon Cu deficiency (Rees et al., 2004). To determine whether BdCOPTs rescue growth defects of the S. cerevisiae ctr1Δctr2Δctr3Δ mutant by facilitating Cu uptake into the cell from the external medium or by vacuolar remobilization, we determined their subcellular localization in yeast cells as well as in brachypodium protoplasts.

For studies in S. cerevisiae, we inserted BdCOPT3, BdCOPT4, and BdCOPT5 cDNAs without the stop codon into the YES3-EGFP-Gate vector to generate translational C-terminal EGFP fusions. We then verified whether the BdCOPT-EGFP constructs were functional by expressing them in the ctr1Δctr2Δctr3Δ mutant and assessing the growth of transformed cells on YPEG media. We found that expression of BdCOPT3-, and BdCOPT4-EGFP fusions in ctr1Δctr2Δctr3Δ cells resulted in growth phenotypes mirroring results of cells expressing un-tagged proteins (Figures 4B,C). However, the BdCOPT5-EGFP construct was unable to rescue growth of ctr1Δctr2Δctr3Δ cells, suggesting that fusing EGFP with BdCOPT5 resulted in the loss of its activity (Figure 4D). Since unfunctional BdCOPT5-EGFP might mislocalize in yeast cells as well as in brachypodium protoplasts, this construct was omitted from subsequent study. We did not analyze the subcellular localization of BdCOPT1 for the same reason. Fluorescent microscopy revealed that EGFP-mediated fluorescence in BdCOPT3-EGFP and BdCOPT4-EGFP expressing cells localized mainly to the cell periphery and the distribution of the EGFP signal was distinct in S. cerevisiae cells expressing the EGFP-only vector (Figure 5). These data suggest that BdCOPT3 and BdCOPT4 localize to the plasma membrane in this heterologous system.

We next analyzed the subcellular localization of BdCOPT3 and BdCOPT4 in brachypodium using transient expression in protoplasts. After establishing a procedure for the isolation of viable protoplasts from brachypodium (Figure 6), we transfected protoplasts with BdCOPT3-EGFP or BdCOPT4-EGFP constructs with C-terminal EGFP fusions expressed from the CaMV promoter of the SAT-N1-EGFP-Gate vector, or with the empty SAT-N1-EGFP-Gate vector. We found that EGFP-mediated fluorescence originating from BdCOPT3-EGFP or BdCOPT4-EGFP constructs was located at the periphery of transfected protoplasts and did not overlap with chlorophyll-mediated autofluorescence (Figures 7A,B and Supplemental Figures 3A,B). Furthermore, fluorescence from BdCOPT3- and BdCOPT4-EGFP constructs was distinct from the fluorescence pattern exhibited by protoplasts transfected with the empty EGFP vector (Figure 7C and Supplemental Figure 3C). We note that internally-localized EGFP-mediated fluorescence is likely an artifact of the degradation of the BdCOPT-EGFPs. We then co-stained protoplasts expressing BdCOPT3- or BdCOPT4-EGFP with the lipophylic dye, FM4-64, which selectively labels the plasma membrane under low-temperature conditions (Vida and Emr, 1995). We found strict co-localization of BdCOPT3- or BdCOPT4-EGFP and FM4-64-mediated fluorescence (Figures 7A,B), suggesting that BdCOPT3 and BdCOPT4 are located at the plasma membrane.

Homo- and heterodimerization of CTR/COPT proteins have been demonstrated in eukaryotes (Lee et al., 2002; De Feo et al., 2007, 2009; Yuan et al., 2010, 2011). Furthermore, although interaction of A. thaliana COPT6 with COPT1 is not required for the ability of these proteins to transport Cu (Jung et al., 2012), the activity of O. sativa CTR/COPT proteins seem to depend on their interactions with each other (Yuan et al., 2011). Therefore, we tested if the CTR/COPT family members in brachypodium would also interact with either themselves or/and with other family members. For this purpose we used the split-ubiquitin-based membrane yeast-two-hybrid (MYTH) approach (Kittanakom et al., 2009). In this system, a modified ubiquitin protein is split into its C- and N-terminal halves (Cub and NubG, respectively), which are fused to membrane-localized bait or prey proteins, respectively. The C-terminus of ubiquitin is attached to an artifical transcription factor, PLV (protein A-LexA-VP16). If bait and prey proteins are oriented in the cytosol and interactions occur, the modified ubiquitin is reconstituted and recognized by ubiquitin-specific proteases, which release PLV from Cub. PLV enters the nucleus to induce expression of lexA-controlled reporter genes ADE2, HIS3, and lacZ (Kittanakom et al., 2009), allowing protein interactions to be assessed by adenine and histidine prototrophy and by the β-galactosidase assay.

We fused Cub-PLV and NubG at the C-terminus of BdCOPT1, BdCOPT3, BdCOPT4, and BdCOPT5 since their predicted topology is consistent with a cytosolic orientation of their C-termini. We then co-expressed BdCOPT proteins in different combinations with themselves or with the empty Cub-PLV vector as a negative control (Figure 8). We included S. cerevisiae co-expressing AtCOPT6-Cub-PLV and AtCOPT6-NubG in our assays as a positive control since AtCOPT6 interacts with itself in the MYTH assay (Jung et al., 2012). To control for false positives, the empty Cub-PLV vector was co-expressed with BdCOPTs fused to NubG. These studies showed that all of the tested BdCOPT proteins interacted in both homo- and heterodimer combinations, regardless of whether interactions were assayed by growth on selective medium or with β-galactosidase (Figure 8). Whether these interactions are required for their ability to transport Cu remains to be elucidated.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.