MtPT5 phosphate transporter is involved in leaf growth and phosphate accumulation of Medicago truncatula

Abstract

Phosphorus (P) is an indispensable mineral nutrient for plant growth and agricultural production. Plants acquire and redistribute inorganic phosphate (Pi) via Pi transporters (PHT1s/PTs). However, apart from MtPT4, functions of the M. truncatula (Medicago truncatula) PHT1s remain unclear. In this study, we evaluated the function of the PHT1 family transporter MtPT5 in M. truncatula. MtPT5 was closely related to AtPHT1; 1 in Arabidopsis (Arabidopsis thaliana) and GmPT7 in soybean (Glycine max). MtPT5 was highly expressed in leaves in addition to roots and nodules. Ectopic expression of MtPT5 complemented the Pi-uptake deficiency of Arabidopsis pht1;1Δ4Δ double mutant, demonstrating the Pi-transport activity of MtPT5 in plants. When overexpressing MtPT5 in M. truncatula, the transgenic plants showed larger leaves, accompanying with higher biomass and Pi enrichment compared with wild type. All these data demonstrate that MtPT5 is important for leaf growth and Pi accumulation of M. truncatula and provides a target for molecular breeding to improve forage productivity.

Keywords: Medicago truncatula; MtPT5; PHT1 transporter; Pi accumulation; leaf growth; phosphate.

Figure 1

Phylogenetic analysis of PHT1s from different species. Phylogenetic tree of PHT1s from Medicago truncatula, Arabidopsis, soybean, maize, and rice. The tree was generated as described in materials and methods. Mt1g074930 (MtPT5) was labeled with a red spot. AtPHT1;1 and GmPT7 were labeled with blue spots. The bar shows 0.05 amino acid substitutions per site.

Figure 2

Expression profiles of MtPTs in M. truncatula. (A) qRT-PCR analysis of MtPT1/2/3 and MtPT5 in different tissues of M. truncatula. Four-day-old wild-type seedlings (R108) were incubated with Sm1021 resuspended in 1/2 Hoagland and then transferred to soil and injected with Sm1021 every 2 days for 1 month. Shoots, roots, and nodules were harvested, respectively, for RNA extraction. Data represent mean ± SE (n = 3). (B) qRT-PCR analysis of MtPT5 in wild-type seedlings (R108) during phosphate starvation. Four-day-old M. truncatula seedlings were transferred to hydroponic solution with Pi (+P) or solution without Pi (−P) for 5 days. The whole seedlings were used for RNA extraction. Data represent mean ± SE (n = 3). ** indicates significant difference at p < 0.01 (Student’s t-test).

Figure 3

Ectopic expression of MtPT5 enhanced Pi acquisition in Arabidopsis. (A) RT-PCR analysis of MtPT5 in wild-type Arabidopsis (Ws), pht1;1Δ4Δ double mutant and 35S:MtPT5/pht1;1Δ4Δ transgenic plants using MtPT5-specific primers. The housekeeping gene EF1α was used as an internal control. (B) Pi content measurement of wild-type Arabidopsis (Ws), pht1;1Δ4Δ double mutant and 35S:MtPT5/pht1;1Δ4Δ transgenic plants. The whole seedlings grown on 1/2 MS for 20 days were collected for Pi extraction. Data represent mean ± SE (n = 3). Different letters indicate significant difference at p < 0.05 (One-way ANOVA, Tukey test). (C) Image of wild-type Arabidopsis (Ws), pht1;1Δ4Δ double mutant and 35S:MtPT5/pht1;1Δ4Δ transgenic plants grown on 1/2 MS containing 200 μM arsenate or without arsenate for 20 days.

Figure 4

Overexpression of MtPT5 promotes leaves growth of M. truncatula. (A) qRT-PCR analysis of MtPT5 in wild-type M. truncatula (R108) and MtPT5-overexpressing plants (35S:MtPT5-1 and 35S:MtPT5-2). Data represent mean ± SE (n = 3). Different letters indicate significant difference at p < 0.05 (One-way ANOVA, Tukey test). (B–D) Phenotypic comparation of wild type (R108) and MtPT5-overexpressing plants. (B) Four-day-old seedlings were transferred to 1/2 Hoagland and grown for 20 days. Inset is leaves detached from the indicated plants. Bars = 1 cm. (C) Plants grown in soil for 3 months. Bar = 5 cm. (D) Top leaflets detached from (C). Bar = 1 cm. (E) Quantification of leaf areas. The fourth leaf of wild type (R108) and MtPT5-overexpressing plants shown in (B) were taken for quantification. Data represent mean ± SE (n = 8). Different letters indicate significant difference at p < 0.05 (One-way ANOVA, Tukey test). (F) Fresh weight of four expanded leaves detached from (B). Data represent mean ± SE (n = 8). Different letters indicate significant difference at p < 0.05 (One-way ANOVA, Tukey test).

Figure 5

Overexpression of MtPT5 enhances Pi accumulation of M. truncatula. (A) Pi content in top leaflets of wild type (R108) and MtPT5-overexpressing plants grown in soil for 3 months. Data represent mean ± SE (n = 3). Different letters indicate significant difference at p < 0.05 (One-way ANOVA, Tukey test). (B) Pi content of wild type (R108) and 35S:MtPT5-overexpressing plants. Four-day-old seedlings were transferred to 1/2 Hoagland and grown for 20 days, then the whole plants were taken for Pi extraction. Data represent mean ± SE (n = 5). Different letters indicate significant difference at p < 0.05 (One-way ANOVA, Tukey test).