An oligopeptide transporter gene family in Arabidopsis

Abstract

We have identified nine oligopeptide transporter (OPT) orthologs (AtOPT1 to AtOPT9) in Arabidopsis. These proteins show significant sequence similarity to OPTs of Candida albicans (CaOpt1p), Schizosaccharomyces pombe (Isp4p), and Saccharomyces cerevisiae (Opt1p and Opt2p). Hydrophilicity plots of the OPTs suggest that they are integral membrane proteins with 12 to 14 transmembrane domains. Sequence comparisons showed that the AtOPTs form a distinct subfamily when compared with the fungal OPTs. Two highly conserved motifs (NPG and KIPPR) were found among all OPT members. The identification of multiple OPTs in Arabidopsis suggests that they may play different functional roles. This idea is supported by the fact that AtOPTs have a distinct, tissue-specific expression pattern. The cDNAs encoding seven of the AtOPTs were cloned into a yeast vector under the control of a constitutive promoter. AtOPT4 expressed in S. cerevisiae mediated the uptake of KLG-[3H]L. Similarly, expression of five of the seven AtOPT proteins expressed in yeast conferred the ability to uptake tetra- and pentapeptides as measured by growth. This study provides new evidence for multiple peptide transporter systems in Arabidopsis, suggesting an important physiological role for small peptides in plants.

Figure 1

Comparison of OPTs. A, Dendogram showing a sequence comparison of the known members of the OPT family. Analysis was performed using the CLUSTAL method in MegAlign (DNASTAR, Madison, WI) using default parameters. Accession numbers are as follows: AtOPT1, AB026659 GI:9758213; AtOPT2, AAB60748 GI:2160185; AtOPT3, Z97341 GI:2244994; AtOPT4, AB008268 GI:9759417; AtOPT5, AL078465 GI:4938497; AtOPT6, AL035602.1 GI:4469024; AtOPT7, AF080119 GI:3600039; AtOPT8, BAB09728.1 GI:9759191; AtOPT9, AB015476 GI:9759190; Opt1p, Z49487; CaOpt1p, U60973; Isp4p, P40900; Opt2p, U25841. The mapped positions of each AtOPT are indicated. B, Hydrophilicity plots of AtOPT1–9 and CaOpt1p as predicted by Kyte and Doolittle (1982). The size (amino acids) of the each protein is shown below the name of the gene. Analysis was performed using Protean (DNASTAR) under default parameters. The bars over each sequence show the location of the two conserved motifs (NPG and KIPPR motifs) (i.e. NPG[P/A]F[N/T/S]XKEH[V/T/A][L/I/V][I/V]I[T/S/V][I/V/M] [F/M][A/S][N/S/A] and K[L/F][G/A][H/M/T]YMK[I/V/L][P/D/S]PR).

Figure 2

Analysis of the OPT sequences. A, Two conserved motifs (NPG and KIPPR motifs) among the OPT members, including fungal OPTs, were determined based on the consensus of their sequences after analysis using the CLUSTAL method in the MegAlign (DNASTAR). Shaded area represents the consensus. B, The putative transmembrane domains of the AtOPTs were determined by the PRED-TMR algorithm. Predicted transmembrane regions of AtOPT1 are shown in bold and the underlined sequences represents the two conserved motifs found in all OPTs.

Figure 3

Tissue-specific mRNA expression of AtOPT1–7. RT-PCR analysis was performed as described in “Materials and Methods” using gene-specific primers. The level of Actin2 mRNA (right panel) was measured as an internal control. F, Flower; L, leaf; S, stem; and R, root.

Figure 4

Peptide growth assays. S. cerevisiae BY4730 strain transformed with pDB20 (vector alone), or expressing CaOpt1p, Opt1p, or AtOPT1–7 were tested for their ability to use Leu-containing peptides (i.e. KLLG, KLGL, KLLLG) to fulfill the auxotrophic requirement for Leu. A, 200 μm KLLLG. The cell number added to each well is shown. B, 200 μm KLGL (left) and 100 μm KLLG (right).

Figure 5

Uptake of KLG-[³H]L by S. cerevisiae BY4730 transformants. Uptake of KLG-[³H]L after 30 min at 30°C or 0°C for cells transformed with pSK104 (AtOPT4), pCaOPT1 (CaOpt1p from C. albicans), and 30°C for cells containing the empty vector pDB20. Uptake is expressed in nanomoles per milligram dry weight on the left y axis, and fraction of uptake as compared with the positive control of CaOpt1p at 30°C on the right y axis. Data shown represent the average of three replicates ± sd.