Functional characterization of 14 Pht1 family genes in yeast and their expressions in response to nutrient starvation in soybean

Abstract

Background: Phosphorus (P) is essential for plant growth and development. Phosphate (Pi) transporter genes in the Pht1 family play important roles in Pi uptake and translocation in plants. Although Pht1 family genes have been well studied in model plants, little is known about their functions in soybean, an important legume crop worldwide.

Principal findings: We identified and isolated a complete set of 14 Pi transporter genes (GmPT1-14) in the soybean genome and categorized them into two subfamilies based on phylogenetic analysis. Then, an experiment to elucidate Pi transport activity of the GmPTs was carried out using a yeast mutant defective in high-affinity Pi transport. Results showed that 12 of the 14 GmPTs were able to complement Pi uptake of the yeast mutant with Km values ranging from 25.7 to 116.3 µM, demonstrating that most of the GmPTs are high-affinity Pi transporters. Further results from qRT-PCR showed that the expressions of the 14 GmPTs differed not only in response to P availability in different tissues, but also to other nutrient stresses, including N, K and Fe deficiency, suggesting that besides functioning in Pi uptake and translocation, GmPTs might be involved in synergistic regulation of mineral nutrient homeostasis in soybean.

Conclusions: The comprehensive analysis of Pi transporter function in yeast and expression responses to nutrition starvation of Pht1 family genes in soybean revealed their involvement in other nutrient homeostasis besides P, which could help to better understand the regulation network among ion homeostasis in plants.

Figure 1. Phylogenetic tree of soybean, Arabidopsis, rice and Medicago plant Pi transporter proteins in Pht1 family.

Transporters and corresponding plant species are as follows: rice (Oryza sativa), OsPT1 through OsPT13 ; Arabidopsis (Arabidopsis thaliana), AtPht1;1 through AtPht1;9 ; Medicago (Medicago truncatula) MtPT1 through MtPT6 and other four PT proteins obtained in Phytozome (http://www.phytozome.net/medicago), soybean (Glycine Max), GmPT1 through GmPT14 (this work).

Figure 2. Complementation of a yeast inorganic phosphate (Pi) transport mutant by GmPTs genes.

Yeast MB192 cells harboring either an empty expression vector (control) or the candidate Pht1 ORF(open reading frame), transformants were grown in YNB medium to an OD600 ≈0.8, then washed by 3% glucose with centrifugation at 1500 g, 4°C, and suspended in phosphate free YNB medium to OD600 ≈1.0. Different number cells (5×10⁵, 5×10⁴, 5×10³) were applied to Pi-limiting medium (20 µM, pH 6.0) then incubated at 30°C for 3 d.

Figure 3. Kinetic analysis of inorganic phosphate (Pi) uptake in yeast.

The non-linear regression of total Pi uptake by strain Yp112-GmPTs versus external Pi concentration at pH 6 were used to estimate the apparent Km value for Pi uptake. All the results were calculated from the three independent experiments.

Figure 4. Spatial Expression pattern analysis for the 14 GmPTs as related to P availability.

Plants were grown on low P (added 5 µM P as KH₂PO_4, open bars) and high P (added 500 µM P as KH₂PO_4, closed bars) conditions. Young leaves (YL), roots (R), stems (S) and flowers (F) were sampled 18 days after treatment initiation, and young pods (P) and seeds (SE) were sampled 29 days after treatment initiation. Each bar is the mean of three biological replications with standard error. Note: different scales are used in the graphs; asterisks indicate significant differences of GmPTs expression in certain tissues under low P and high P conditions in t-tests.

Figure 5. Expression of P, N, K or Fe responsive genes to different nutrient stresses.

14-day old soybean seedlings were treated with N (−N), K (−K) and Fe (−Fe) deficiencies (see Experimental procedures for details). Seedlings grown under normal solution were used as controls (CK, added 500 µM P as KH₂PO₄). The expression levels in shoots and roots were analyzed by quantitative real-time PCR. Soybean gene PLDZ (Glyma20g38200) was used as low P responsive gene (A), NiR (Glyma02g14910) for low N treatment (B), HAK (Glyma3g42480) for low potassium treatment (C) and IRT (Glyma07g34930) for low Fe treatment (D), respectively. Each bar was the mean of three biological replications with standard error.

Figure 6. Responses of GmPTs to different nutrient stresses.

Ten-day old soybean seedlings were treated with N (−N), K (−K) and Fe (−Fe) deficiencies (see Experimental procedures for details). Seedlings grown under normal solution were used as controls (CK, added 500 µM P as KH₂PO₄). Asterisks indicated the significant differences of GmPTs expression between nutrients deficient stresses and normal conditions in Student’s t-tests.