Regulated expression of Arabidopsis phosphate transporters

Abstract

Phosphorus deficiency is one of the major abiotic stresses affecting plant growth. Plants respond to the persistent deficiency of phosphate (Pi) by coordinating the expression of genes involved in alleviation of the stress. The high-affinity Pi transporters are among the major molecular determinants that are activated during Pi stress. In this study, using three reporter genes (green fluorescent protein, luciferase, and beta-glucuronidase) regulated by two Pi transporter promoters, we have carried out an extensive analysis of transcriptional and spatial regulation of gene expression. Activation of the genes was rapid, repressible, and specific in response to changes in Pi availability. The phytohormones auxin and cytokinin suppressed the expression of the reporter gene driven by the AtPT1 promoter, and that of the native gene, suggesting that hormones may be involved in regulation of some component(s) of Pi starvation response pathway. These studies also provide molecular evidence for a potential role of high-affinity Pi transporters in mobilizing Pi into reproductive organs. The results suggest that members of the Pi transporter family may have similar but nonredundant functions in plants.

Figure 1

Pi starvation-induced expression of reporter genes. A, Transgenic Arabidopsis expressing the reporter genes GUS (1 and 2) and GFP (3 and 4) under the regulation of AtPT1 promoter are shown. 1 and 3, Expression of reporter genes under Pi sufficiency conditions. 2 and 4, Expression under Pi deficiency conditions. B, Transgenic Arabidopsis expressing the reporter gene GUS (1 and 2) or LUC (5 and 6) under the regulation of AtPT2 promoter are shown. 1 and 5, Absence of expression of GUS and LUC in plants grown under Pi sufficiency. Expression of GUS and LUC under Pi-starved conditions is shown in 2 and 6, respectively. 3 and 4, Pi-sufficient and -deficient plants used for monitoring LUC expression in roots.

Figure 2

Effect of Pi concentration on gene expression. A, Seven-day-old seedlings of AtPT2-LUC plants were transferred to Murashige and Skoog media containing varying concentrations of Pi (0, 5, 10, 25, 50, 125, 250, 500, and 1,250 μm) for 5 d. Plants were harvested for measuring the LUC activity and RNA isolation. B, Seven-day-old seedlings of AtPT2-GUS plants were grown in the presence of different concentrations of Pi as indicated for 5 d and utilized for GUS reporter expression analysis. C, Northern analysis of total RNA from AtPT2-LUC plants supplemented with different concentrations of Pi. Ten micrograms of total RNA was electrophoretically separated on denaturing formaldehyde agarose gels and blotted on to nylon membranes. The membranes were probed with ³²P-labeled AtPT1 and AtPT2 cDNAs. The ethidium bromide-stained gel picture shows uniform loading of RNA samples.

Figure 3

Activity of AtPT1 promoter in response to Pi starvation. Seven-day-old AtPT1-GUS/GFP expressing plants were transferred to liquid Murashige and Skoog medium with (1,250 μm) and without (0 μm) Pi for 5 d. Soluble protein was extracted from the Pi-treated plants to determine GUS activity. The enzyme activity was measured using 4-methyumbelliferyl-β-d GlcUA as the substrate. Error bars show sd. B, Northern-blot analysis of total RNA isolated from Arabidopsis plants grown in the presence (P+) and absence (P−) of Pi for 5 d. The blot was probed with ³²P-labeled AtPT1. The ethidium bromide-stained gel shows uniform loading and integrity of RNA.

Figure 4

Temporal expression of Pi transporters. A, Rapid induction of AtPT2 promoter-driven expression of LUC during Pi deficiency was monitored in the transgenic plants. Seven-day-old seedlings of AtPT2-LUC plants were transferred to Murashige and Skoog medium without Pi. The seedlings were removed at different times after Pi starvation to measure reporter gene activity and for northern analysis of transcripts. B, Total RNA from the plants harvested at different times of Pi deficiency was separated on denaturing formaldehyde agarose gels and blotted onto nylon membranes. The membranes were probed with ³²P-labeled cDNA fragments of AtPT1, AtPT2, and LUC. C, Reversibility of induction of genes was studied by resupplying Pi to Pi-starved plants. The LUC expressing plants (AtPT2-LUC) were starved for Pi for 5 d and then transferred to Murashige and Skoog medium containing sufficient Pi (1,250 μm). Other samples of seedlings were grown continuously under Pi deficiency. Plants were harvested at different time periods (12 h, and 1, 2, 3, and 4 d) after transfer, and analyzed for reporter gene activity and isolation of RNA. DR, Days after replenishment with Pi; DP, days plants continued to be grown in the absence of Pi. D, Northern blot showing the expression of AtPT1, AtPT2, and LUC in plants subjected to Pi replenishment experiments. The nylon membranes containing the RNA were probed with ³²P-labeled cDNAs.

Figure 5

Pi transporter promoter-mediated expression of reporter genes in Arabidopsis root. A, AtPT1 promoter-mediated expression of GUS (1–3) and GFP (4–6) in Arabidopsis roots and root hairs (3 and 6) are shown. Stronger GUS activity can be observed in newly formed branches, whereas the intensity of GUS staining decreased in the primary root (2). Lack of GUS expression in the tips of primary and lateral roots of AtPT1-GUS plants is clearly depicted (1 and 2). AtPT2 promoter-driven expression of GUS in all parts of the roots, including root tips and root hairs, is shown in 7 through 9. B, Expression of the reporter gene activity was examined in thin sections of roots of transgenic plants. The Pi-deficient plant roots expressing GUS were allowed to develop blue color in the presence of X-Gluc. The root segments were fixed and embedded in Technovit resin and 8-μm sections were cut and placed on a slide. The sections were photographed under a microscope (Olympus Corporation, Lake Success, NY). Top, GUS staining of the transverse sections of roots; bottom, cross-sectional view. 1 through 4, Expression of the AtPT1-GUS in the root tip (1 and 3) and the differentiated region of a root (2 and 4). Similarly, the AtPT2-GUS expression can be seen in the root tip (5 and 7) and the differentiated region (6 and 8).

Figure 6

A, AtPT2 promoter-mediated expression of reporter genes in flowers and fruits of Pi-starved Arabidopsis. Expression of GUS (1 and 3) and LUC (2 and 4) in flowers (1 and 2) and fruits (3 and 4) was examined in Pi-starved plants. The reporter gene expression is confined to the silique and stalk junctions in fruits (3 and 4). Transverse (5) and cross-sectional (6 and 7) views of the silique and stalk junction clearly show the GUS expression. Strong expression of reporter gene is observed in outer cell layers (reminiscent of nectaries) and some cells in tracheary elements and vascular tissues. Cross-sectional view of the fruit stalk (8) showing the expression of GUS in some cells of cortical region. B, Heterologus expression of reporter genes in tobacco (Nicotiana tabacum). Expression of LUC (1) and GUS (2) under the regulation of AtPT2 promoter in tobacco roots. Longitudinal (3) and cross-sectional (4) view of the root showing the GUS expression.

Figure 7

Effect of hormones or hormone inhibitors on GUS activity and expression of Pi starvation-responsive genes. Seven-day-old seedlings of AtPT1-GUS/GFP and AtPT2-GUS were transferred to Murashige and Skoog medium with (P+) and without (P−) Pi supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 0.5 μm), naphthalene acetic acid (0.5 μm), kinetin (Ki; 0.1, 1.0 and 10 μm), 6-benzyleaminopurine (1.0 μm), 1-aminocyclopropane-1-carboxylic acid (ACC; 50 μm), 2,3,5-triiodobenzoic acid (1.0 μm), naphthalmic acid (1.0 μm), or α-2-aminoethoxyvinyl Gly (AVG; 10 μm). Seedlings were harvested after 48 h of treatment for enzyme assay and RNA isolation. Effect of hormones and inhibitors of hormones on AtPT1 (A and D) and AtPT2 (B and E) promoter-driven expression of GUS under Pi-sufficient (white bars) and -deficient (black bars) conditions. Error bars represent sd. Northern analysis of total RNA from AtPT2-GUS plants treated with different hormones and hormone inhibitors (C and F). Fifteen micrograms of total RNA was transferred to nylon membranes. The membranes were hybridized with ³²P-labeled AtPT1, AtPT2, At4, PAP, RNase2, and tubulin cDNA fragments.