Reducing the genetic redundancy of Arabidopsis PHOSPHATE TRANSPORTER1 transporters to study phosphate uptake and signaling

Abstract

Arabidopsis (Arabidopsis thaliana) absorbs inorganic phosphate (Pi) from the soil through an active transport process mediated by the nine members of the PHOSPHATE TRANSPORTER1 (PHT1) family. These proteins share a high level of similarity (greater than 61%), with overlapping expression patterns. The resulting genetic and functional redundancy prevents the analysis of their specific roles. To overcome this difficulty, our approach combined several mutations with gene silencing to inactivate multiple members of the PHT1 family, including a cluster of genes localized on chromosome 5 (PHT1;1, PHT1;2, and PHT1;3). Physiological analyses of these lines established that these three genes, along with PHT1;4, are the main contributors to Pi uptake. Furthermore, PHT1;1 plays an important role in translocation from roots to leaves in high phosphate conditions. These genetic tools also revealed that some PHT1 transporters likely exhibit a dual affinity for phosphate, suggesting that their activity is posttranslationally controlled. These lines display significant phosphate deficiency-related phenotypes (e.g. biomass and yield) due to a massive (80%-96%) reduction in phosphate uptake activities. These defects limited the amount of internal Pi pool, inducing compensatory mechanisms triggered by the systemic Pi starvation response. Such reactions have been uncoupled from PHT1 activity, suggesting that systemic Pi sensing is most probably acting downstream of PHT1.

Figure 1.

Characterization of the triple mutant. A, Western-blot analysis of PHT1;1, PHT1;2, and PHT1;3 on root membrane protein extracts. No PHT1;2 or PHT1;3 proteins could be detected in pht1;1 grown in +P. Lanes were loaded with 10 or 20 μg of root proteins extracted from plants grown in –P or +P medium, as detailed in the figure. A loading control western blot, using the anti-Plasma Membrane Intrinsic Protein1 (PIP1) antibody, is shown at bottom. B, ³³P uptake capacity in +P conditions. C, ³³P partitioning (leaves [L] per total P) in +P conditions. For B and C, each bar corresponds to 22 to 24 individual plants. All experiments were performed at least twice. Error bars represent se. WT, Wild type; L+R, leaves + roots.

Figure 2.

Characterization of the quadruple and quintuple mutants. A, Pi content in leaves (gray bars) and roots (white bars) of plantlets grown in +P at 11 dpg, including a wild-type (WT) control grown in –P. sds are shown. B, Western-blot analysis of PHT1;1, PHT1;2, and PHT1;3 on root membrane protein extracts. No residual protein was detected in the mut5 line. The first lane was loaded with 10 μL of Bio-Rad Precision Plus Protein Standards (Std); the following lanes were loaded with 0.5 to 20 μg of root proteins extracted from plants grown in –P medium, as detailed in the figure. A loading control western blot, using the anti-PIP1 antibody, is shown at bottom. C, ³³P uptake capacity is strongly affected in mut4 and mut5 lines. Pi uptake capacity was calculated as nmol Pi h^–1 cm^–1 root. Pi uptake was performed with 12-dpg plantlets grown in +P (gray bars) or in –P media (white bars). Each bar corresponds to 17 to 24 individual plants. Error bars represent se. Values above each bar correspond to Pi uptake percentage compared with the wild type. Data shown are from a representative experiment; experiments were performed in triplicate. FW, Fresh weight.

Figure 3.

Quadruple and quintuple mutant lines show transcriptional induction of PSI and PHT1 markers in Pi-sufficient growth conditions. A, qRT-PCR of several members of the PHT1 family and several PSI genes in two independent mut4 lines. B, qRT-PCR of several members of the PHT1 family and several PSI genes in two independent mut5 lines. The wild type (WT) grown on +P and on –P have been included as controls. Corresponding loci numbers and primer sequences are provided in Supplemental Table S2. Biological triplicates were performed, and all samples were analyzed with technical triplicates. sds are shown.

Figure 4.

³³P uptake kinetics demonstrates that PHT1-depleted lines are affected both in low- and high-affinity transport. Eadie and Hofstee representation of the ³³P uptake kinetic parameters. The experimental points are the mean ± sd of a representative experiment with at least 11 individual plantlets for each genotype and each condition. The dotted line insert is a magnified view of the dotted line selection in the main graph. High signifies high-affinity transport (2–100 μm Pi), and Low represents low-affinity transport (0.2–2 mm Pi). WT, Wild type.

Figure 5.

Biomass and roots-to-leaves ratio of mut4 and mut5 lines. A, Average biomass of leaves (gray bars) and roots (white bars) expressed as mg of fresh weight (FW) in 12-dpg plantlets grown in –P conditions (n = 30–80 plantlets). B, Average biomass of leaves (gray bars) and roots (white bars) expressed as mg of fresh weight in 12-dpg plantlets grown in +P conditions (n = 15–30 plantlets). C, Ratio of roots (R) to leaves (L; fresh weight biomass) in –P (white), +P (pale gray), and VHPi (10 mm ; dark gray) conditions. All experiments were reproduced at least twice. For A and B, bilateral Student’s t tests were conducted to assess statistical differences between the wild type (WT) and the other genotypes, comparing leaf biomass to wild-type leaf biomass and root biomass to wild-type root biomass. In A, the single asterisk indicates P < 0.001. In B, the single asterisk indicates P < 0.05.

Figure 6.

Physiological characterization of mut4 and mut5 lines on VHPi. In vitro phenotyping of the lines used in this study in +P (A) and VHPi (10 mm Pi; B) at 8 dpg. C, Average biomass of leaves (left) and roots (right) in +P (white bars), 2 mm Pi (light-gray bars), and 10 mm Pi (dark-gray bars) at 12 dpg. Bilateral Student’s t tests were conducted to assess statistical differences between the wild type (WT) and the other genotypes for every growing condition and comparing leaves to wild-type leaves biomass and roots to wild-type root biomass. The single asterisk indicates P < 0.5; the double asterisk indicates P < 0.001, and the triple asterisk indicates P < 0.0001. D, Pi content in leaves (gray bars) and roots (white bars) of wild-type, mut4, and mut5 lines in +P, 2 mm Pi, and VHPi at 12 dpg. E, ³³P uptake capacity of the wild type and mut5-2 in –P, +P, and VHPi growth/incubation conditions. F, ³³P partitioning of the plants from E. For E and F, 20 to 24 plants were used for each genotype/condition, and plantlets were analyzed at 9 dpg. All experiments were repeated at least twice. FW, Fresh weight.

Figure 7.

³³P uptake capacity in VHPi is mainly a passive process. A, Wild-type (WT) uptake capacity is reduced by CCCP addition, except in VHPi conditions. Wild-type plants were grown for 10 d in –P, +P, and VHPi before incubation in the corresponding media in control (white bars) or +10 μm CCCP (gray bars) conditions. B, In VHPi conditions, mut5 lines show the same uptake capacity as the wild type, both in the control (white bars) and with +CCCP addition (gray bars). Pi uptake capacity was calculated as nmol Pi h^–1 cm^–1 root. Data shown are from a representative experiment; experiments were performed in either duplicate or triplicate. Each bar corresponds to 20 to 24 plants. Error bars represent se.

Figure 8.

VHPi conditions restore transcriptional repression of PHT1 and PSI markers in mut5 lines. qRT-PCR of several members of the PHT1 family and several PSI genes in the wild type (WT), a mut4 line, and two independent mut5 lines. Plantlets were grown on +P as well as 2 and 10 mm Pi media (VHPi) for 11 d before RNA extraction from roots. Corresponding loci numbers and primer sequences are provided in Supplemental Table S2. Biological triplicates were performed, and all samples were analyzed with technical triplicates. sds are shown.