Overexpression of an Arabidopsis thaliana high-affinity phosphate transporter gene in tobacco cultured cells enhances cell growth under phosphate-limited conditions

Abstract

A higher plant homologue to the high-affinity phosphate transporter gene of yeast (Saccharomyces cerevisiae) PHO84 was isolated from Arabidopsis thaliana. Expression of the Arabidopsis gene PHT1 at high levels in tobacco-cultured cells increased the rate of phosphate uptake. The uptake activity attributable to the transgene was inhibited by protonophores, suggesting an H+ cotransport mechanism of phosphate uptake, and had a Km of 3.1 microM which is within limits characteristic of high-affinity transport mechanisms. These results indicate that PHT1 encodes a high-affinity phosphate transporter. The transgenic cells exhibited increased biomass production when the supply of phosphate was limited, establishing gene engineering of phosphate transport as one approach toward enhancing plant cell growth.

Figure 1

Structure of the PHT1 gene and the construct for plant transformation. (Upper) The structure of the PHT1 gene is shown. Open boxes, exon sequences; closed boxes, intron sequences; S, SacI; E, EcoRI; A, ApaI; B, BamHI; H, HindIII. The positions of the translation start codon (ATG) and the translation termination codon (TAA) are shown with arrowheads. Note that the first intron (236 bp) locates in the 5′-untranslational region. (Lower) To introduce the PHT1 gene into tobacco cells by particle bombardment, the SacI–HindIII fragment of PHT1 was inserted between the cauliflower mosaic virus 35S promoter (CaMV 35S) and the nopalin synthase gene terminator (tnos) in a plant vector pMi12, named p35SPHT1. pMi12 carries the neomycin phosphotransferase II (NPTII) gene, driven by the nopalin synthase gene promoter (pnos), for selection of transgenic cells by kanamycin.

Figure 2

Phosphate uptake of transgenic tobacco cells. Inorganic phosphate uptake rates of suspension-cultured cells carrying the A. thaliana phosphate transporter construct p35SPHT1 (filled bar) and the control construct pMi12 (open bar) were measured. Data are the mean ± SD from nine experiments. The amounts of PHT1 mRNA and the 28S rRNA on a Northern blot of each cell line (C11, P38, P11, P2, P10, P27, and P20) are shown under the histogram.

Figure 3

Phosphate uptake in transgenic tobacco cultured cells carrying the Arabidopsis gene PHT1. Phosphate uptake rates (V; n mol/h per g fwt) in a transgenic cell line carrying p35SPHT1 (P11) (•) and a control cell line carrying pMi12 (C11) (○) were determined by the depletion-curve method (23). [S], phosphate concentration (μM).

Figure 4

pH-dependence of phosphate uptake of transgenic tobacco cells. Cultured cells carrying either p35SPHT1 (•) or the vector pMi12 alone (○) were washed four times with phosphate-free medium (see Materials and Methods) of different pH (adjusted by Tris). Just before the phosphate uptake experiments, pH in each medium was measured. Data are the mean ± SD from three experiments.

Figure 5

The chromosomal position of the PHT1 gene. The recombinant inbred lines of ecotypes Columbia and Landsberg erecta (31) and restriction fragment length polymorphism markers (31, 32) were used for mapping of the gene. Segregation data from 100 chromosomes were analyzed with the computer program mapmaker (version 2.0).