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. 2014 May 30:5:236.
doi: 10.3389/fpls.2014.00236. eCollection 2014.

Brachypodium distachyon as a model system for studies of copper transport in cereal crops

Ha-Il Jung  1 , Sheena R Gayomba  1 , Jiapei Yan  1 , Olena K Vatamaniuk  1
Affiliations

Brachypodium distachyon as a model system for studies of copper transport in cereal crops

Ha-Il Jung et al. Front Plant Sci. .

Abstract

Copper (Cu) is an essential micronutrient that performs a remarkable array of functions in plants including photosynthesis, cell wall remodeling, flowering, and seed set. Of the world's major cereal crops, wheat, barley, and oat are the most sensitive to Cu deficiency. Cu deficient soils include alkaline soils, which occupy approximately 30% of the world's arable lands, and organic soils that occupy an estimated 19% of arable land in Europe. We used Brachypodium distachyon (brachypodium) as a proxy for wheat and other grain cereals to initiate analyses of the molecular mechanisms underlying their increased susceptibility to Cu deficiency. In this report, we focus on members of the CTR/COPT family of Cu transporters because their homologs in A. thaliana are transcriptionally upregulated in Cu-limited conditions and are involved either in Cu uptake from soils into epidermal cells in the root, or long-distance transport and distribution of Cu in photosynthetic tissues. We found that of five COPT proteins in brachypodium, BdCOPT3, and BdCOPT4 localize to the plasma membrane and are transcriptionally upregulated in roots and leaves by Cu deficiency. We also found that BdCOPT3, BdCOPT4, and BdCOPT5 confer low affinity Cu transport, in contrast to their counterparts in A. thaliana that confer high affinity Cu transport. These data suggest that increased sensitivity to Cu deficiency in some grass species may arise from lower efficiency and, possibly, other properties of components of Cu uptake and tissue partitioning systems and reinforce the importance of using brachypodium as a model for the comprehensive analyses of Cu homeostasis in cereal crops.

Keywords: Brachypodium; CTR/COPT transporters; copper homeostasis; copper transport; wheat.

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Figures

Figure 1
Figure 1
Gene structure of BdCOPT2 and phylogenetic analysis of the brachypodium COPT family. (A) Gene structure of BdCOPT2. Note the presence of introns, which are absent in other plant CTR/COPT members. Scale bar = 500 bp. (B) A phylogenetic tree of CTR/COPT members of A. thaliana (designated as AtCOPT1-6), O. sativa (designated as OsCOPT1-7), brachypodium (designated as BdCOPT1-5), and S. cerevisiae (designated as Ctr1p-CTR3p). The A. thaliana protein, IRT1, is included as an outgroup.
Figure 2
Figure 2
A diagram showing conserved motifs within the primary sequence of BdCOPT proteins and their homologs in A. thaliana, AtCOPT1 and AtCOPT6. Topology predictions are based on the TMHMM software, version 1.0. Shown are methionine-rich motifs within the predicted N-terminal domain (Mets-motifs, gray bars), transmembrane domains (TM1, TM2, and TM3; black bars) and cysteine-rich motifs within the predicted C-terminal domain (white bars). Asterisks “***” and “**” indicate the location of conserved MXXXM and GXXXG motifs. Note that BdCOPT3 has a unique structure of two transmembrane domains and that BdCOPT3 and BdCOPT4 have cysteine-rich domains in the C-terminal domain.
Figure 3
Figure 3
Quantitative real-time (qRT)-PCR analysis of the effect of Cu on expression COPT1 through 5 in roots (A), young leaves (B), and older leaves (C) of brachypodium. For all treatments, 7-day-old wild-type seedlings were transferred to hydroponic solution and were grown for 18 days in medium that, in addition to macro- and micronutrients, contained either 0.25 μM CuSO4 (control conditions), 0 μM CuSO4 (Cu limited), 0 μM CuSO4 + 500 μM BCS (Cu deficiency), or 3 μM CuSO4 (Cu toxicity). Error bars show SE (n = 9). Differences of the mean values between control and treated plants are indicated as *p ≤ 0.05 or **p ≤ 0.001. Results are presented relative to the expression of genes under control conditions.
Figure 4
Figure 4
BdCOPT3, BdCOPT4, and BdCOPT5 rescue the growth defect of the S. cerevisiae ctr1Δctr2Δctr3Δ triple mutant on ethanol/glycerol medium (YPEG). The ctr1Δctr2Δctr3Δ mutant was transformed with the YES3-Gate vector harboring BdCOPT1 (A), BdCOPT3 (B), BdCOPT4 (C), and BdCOPT5 (D) along with corresponding EGFP-fusions and spotted onto YPEG plates supplemented with the indicated concentrations of CuSO4. As negative controls, the ctr1Δctr2Δctr3Δ mutant strain was transformed with the empty YES3-Gate (ctr1,2,3Δ) or empty YES3-EGFP-Gate vector (EGFP). The isogenic wild-type, SEY6210, transformed with the empty YES3-Gate vector (Wt) and ctr1Δctr2Δctr3Δ cells transformed with YES3-Gate harboring the A. thaliana COPT6 (AtCOPT6) cDNA insert were used as positive controls.
Figure 5
Figure 5
Subcellular localization of BdCOPT3-EGFP (A) or BdCOPT4-EGFP (B) fusions or EGFP (C) in S. cerevisiae ctr1Δctr2Δctr3Δ cells. Superimposed images (Overlay) from differential interference contrast microscopy (DIC) and EGFP-mediated fluorescence (EGFP) show that BdCOPT3 and BdCOPT4 localize to the plasma membrane and that the pattern of fluorescence of EGFP-fused proteins is distinct from EGFP.
Figure 6
Figure 6
Isolation of protoplasts from brachypodium. Hydroponically grown 25-day-old plants (A) were used for the isolation of protoplasts from leaf tissue. Chopped brachypodium leaves in filter-sterilized TVL solution are shown in (B). Enzymatic digestion of the cell wall and fractionation by sucrose density gradient yielded protoplasts at the interface of the enzyme solution and W5 solution (C, black arrow). Protoplasts were collected and purified from sucrose density gradient solution (D) and visualized under microscopy using bright-field filter sets (E). In our method, 0.2 g of leaf tissue from 25-day-old seedlings yields 5 × 106–107 protoplasts. Close-up of brachypodium protoplasts through bright-field (F) and FITC (G) filter sets to assess protoplast viability after staining with the membrane-permeable non-fluorescent dye, fluorescein diacetate. After diffusion into viable protoplasts fluorescein diacetate is hydrolyzed into a polar compound, causing the cytosoplasm of the cell to fluoresce under the FITC filter set. Scale bar = 20 μm.
Figure 7
Figure 7
Subcellular localization of BdCOPT3 and BdCOPT4 in brachypodium protoplasts. Protoplasts isolated from leaves of 25-day-old plants were transfected with BdCOPT3-EGFP (A) or BdCOPT4-EGFP (B) constructs or the empty SAT6-N1-EGFP vector (C) and co-stained with the plasma-membrane dye, FM4-64. EGFP-mediated fluorescence derived from BdCOPT3-EGFP (COPT3) or BdCOPT4-EGFP, (COPT4), or from EGFP of the SAT6-N1-EGFP vector (EGFP) was detected using the FITC filter set while FM4-64 (FM4-64) and chlorophyll autofluorescence (Chl) were visualized using the Rhodamine filter set of an Axio Imager M2 microscope equipped with a motorized Z-drive (Zeiss). Images collected from FITC and Rhodamine filter sets were overlaid (Overlay) to show the plasma membrane subcellular localization of Cu transporters. Scale bar = 10 μm.
Figure 8
Figure 8
Analyses of protein-protein interactions of brachypodium CTR/COPT transporters using the split-ubiquitin membrane yeast two-hybrid system (MYTH). Shown are yeast cells co-expressing NubG constructs fused with BdCOPT1, BdCOPT3, BdCOPT4, or BdCOPT5 cDNA (BdCOPT1-, BdCOPT3-, BdCOPT4-, BdCOPT5-NubG) and a Cub-PLV construct lacking a cDNA insert (Cub-PLV) or Cub-PLV fused to BdCOPT1-, BdCOPT3-, BdCOPT4-, BdCOPT5 (BdCOPT1-, BdCOPT3-, BdCOPT4-, BdCOPT5-Cub). Interactions were visualized by colony formation on selective media SC medium (SC) or as blue colonies in a ß-galactosidase assay (SC + Ade + His + X-gal). Growth was monitored for 2 days under conditions indicated below each panel. Interactions of AtCOPT6 with itself (AtCOPT6-NubG + AtCOPT6-Cub), or lack of interactions between AtCOPT6-Cub-PLV and the empty NubG vector (NubG + AtCOPT6-Cub) were used as controls and are indicated in red. Shown are representative results of at least three biological replicates. SC, synthetic complete medium; Ade, adenine; His, histidine; X-gal, bromo-chloro-indolylgalactopyranoside.
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