Figure 1.
Sequence alignment among the TcOPT3, AtOPT3 ATOPT3, ZmGT and BjGT1. CLUSTAL W (version 1.8) alignment of deduced amino acid sequences from the OPTs. Amino acids are numbered from the initiator ATG. Black-shaded areas represent the consensus, dark-gray-shaded areas represent identical amino acids, and light-gray-shaded areas represent similar amino acids. The putative transmembrane (TM) domains of the TcOPT3 were determined by the TMHMM algorithm. The predicted transmembrane membrane spanning domains are shown as lines above the sequences, and numbered TM I–TM XIV respectively. The bars under the sequence show the location of the two conserved motifs (NPG and KIPPR motifs). Tc, T. caerulescens; At, Arabidopsis thaliana; Als, Arabidopsis lyrata subsp.; Zm, Zea mays; Bj, Brassica juncea. Accession numbers are: HQ699884 (TcOPT3), NP_567493 (AtOPT3), XP_002868139 (AlsOPT3), ACL82964 (ZmGT), CAD91127.1 (BjGT1).
Figure 2.
Phylogenetic tree of OPT gene transporters based on the amino acid sequences.
Dendogram showing sequence comparisons of several known members of the PT family from different species. Analysis was performed using the CLUSTAL X method in MEGA (4.0) using Neighbor-Joining method (Tamura K et al., 2007). Accession numbers are as follows: AtOPT1, NP_200404.1 GI: 15241078; AtOPT2, NP_172464.1 GI15218331; AtOPT3, NP_567493.5 GI:240255930; AtOPT4, NP_201246.1 GI:15237689; AtOPT5, NP_194389.1 GI:15236800; AtOPT6, NP_194503.1 GI:15234254; AtOPT7, NP_192815.1 GI:15236912; AtOPT8, NP_564525.1 GI:18402162; AtOPT9, NP_200163.1 GI:15238761; AlOPT3, XP_002868139.1 GI:297800510; AtYSL1, NP_567694.2 GI:79484897; AtYSL2, NP_197826.2 GI:79518939; AtYSL3, NP_200167.2 GI:145359208; AtYSL4, NP_198916.2 GI:42568235; AtYSL5, NP_566584.1 GI:18401590; AtYSL6, NP_566806.1 GI:18405202; AtYSL7, NP_176750.1 GI:15218799; AtYSL8, NP_564525.1 GI:18402162; TcYSL1, ABB76761.1 GI:82468791; TcYSL2, ABB76762.1 GI:82468793; TcYSL3, ABB76763.1 GI:82468795; CaOPT1, AAB69628.1 GI:2367386; CaOPT3, ABD17824.1 GI:87045965; ScOPT1, NP_012323.1 GI:6322249; ZmGT, ACL82964.1 GI:220901863 BjGT1, CAD91127.1 GI:30722286.
Figure 3.
Tissue-specific analysis of the TcOPT3.
Real-time RT-PCR expression analysis of the TcOPT3 gene expression in roots (R), leaves (L) and stems (S). The ΔCp values were calculated as follows: CP of target gene (TcOPT3) – CP of constitutive control gene (ubiquitin-conjugating enzyme), where the CP value is the fractional cycle number of crossing point (CP). The ΔCP values represent the mean of three technical replicates (±SD) of one experiment representative of three independent experiments. Relative transcript levels (RTL) were calculated as follows: RTL = 2−ΔCP.
Figure 4.
Localization of TcOPT3 expression by in situ hybridization.
(A,C,E,G,I) represents hybridization with TcOPT3 antisense probe. (B,D,F,H,J) shows hybridization with the sense probe (negative control). In situ hybridization of the sense and antisense TcOPT3 probes to sections of Thlaspi caerulescens root tissues (A) to (D), stem tissues(E,F,I,J) and leaf tissues(G,H). Abbreviation: c, cortex; ep, epidermis; p, pericycle; ph, phloem; rh, root hair; x, xylem; cb, cambium; ve, vein; me, mesophyll; vc, vascular cambium.
Figure 5.
Effect of element deficiency on mRNA expression of the TcOPT3 gene.
Real-time RT-PCR expression analysis of the TcOPT3 gene expression in roots (R), leaves (L) and stems (S) with the treatment of Fe or Zn deficient for 1, 2, 4 days. The ΔCp values were calculated as follows: CP of target gene (TcOPT3) – CP of constitutive control gene (ubiquitin-conjugating enzyme), where the CP value is the fractional cycle number of crossing point (CP). The ΔCP values represent the mean of three technical replicates (±SD) of one experiment representative of three independent experiments. Relative transcript levels (RTL) were calculated as follows: RTL = 2−ΔCP.
Figure 6.
Sub-cellular localization of TcOPT3 protein.
Onion epidermal cells transiently co-transformed with TcOPT3::GFP and pm-rk (Plasma membrane marker). (A) Fluorescence image of epidermal cell expressing the p35S::EGFP fusion protein. (B) Fluorescence image of epidermal cell expressing the pm-rk. (C) Merged fluorescence image of epidermal cell expressing the p35S-TcOPT3::EGFP fusion protein and pm-rk marker.
Figure 7.
Complementation of the fet3fet4 and ZHY3 (zrt1ztr2) yeasts mutant by T. caerulescens cDNAs.
Yeast strains defective in iron uptake (fet3fet4) and zinc uptake (zrt1ztr2) were transformed with pFL61 (empty vector) and pFL61-TcOPT3. Serial dilutions of yeast cells were dropped onto a low-zinc medium (LZM) supplemented with 50 µM ZnSO4 (A) and a low-iron medium (LIM) supplemented with 10 µM FeCl3 (B) assayed for growth on SD-ura plates. The entire experiment was performed twice.
Figure 8.
Growth of the wild-type (DY1455) and TcOPT3-transformed yeast cells under different metal supplies.
Yeast cells were grown to an OD600 of 1.0, then supplemented with 50 µM FeCl3, 50 µM ZnSO4, 20 µM CdCl2 or 50 µM CuSO4 respectively. Data are the means ± SE per experiment (n = 3), P<0.05 by Student’s t-test.
Figure 9.
Heavy metal accumulation of wild-type (DY1455) and TcOPT3-transformed yeast cells.
Zn, Fe, Ni, Cd and Cu accumulation in yeast transformants. Metal accumulation was conducted in liquid SD media supplemented with 50 µM FeCl3, 50 µM ZnSO4, 20 µM CdCl2, or 50 µM CuSO4 respectively. Data are the means ± SE per experiment (n = 3), P<0.05 by Student’s t-test.
Table 1.
Primers used for PCR amplification of in TcOPT3 cDNAs, 5′ and 3′ RACE, qRealTime-PCR, and plasmid constructions in order of their first mention in “Materials and Methods”.